Reagents and methods for detecting infectious diseases

ABSTRACT

Provided herein are surface enhanced Raman scattering (SERS)-active reagents and methods for detecting one or more analyte in a sample. Said SERS-active reagents are adaptable, sensitive, and easy-to-use in the diagnosis of infectious diseases in a patient, or the detection of toxins, bacteria, viruses, pathogens, hormones, cytokines, antigens, antibodies or illicit drugs in a biological sample. Such methods may be handled by police, soldiers, or health care workers in the field, and do not require specialized training.

CROSS REFERENCE TO RELATED APPLICATIONS

This application is a continuation of U.S. patent application Ser. No.15/552,196, filed Aug. 18, 2017, which is the U.S. national stage ofInternational Application PCT/US16/18600, filed Feb. 19, 2016, whichclaims priority to U.S. Provisional Application No: 62/118,453, filedFeb. 19, 2015, U.S. Provisional Application No. 62/263,995, filed Dec.7, 2015, and U.S. Provisional Application No: 62/264,088, filed Dec. 7,2015, the contents of each are incorporated herein by reference in theirentirety.

BACKGROUND

The present invention is directed to products for and methods ofdetecting analytes bound by functionalized aptamers usingsurface-enhanced Raman scattering (SERS). The system is composed ofthree basic parts: a SERS-active surface, an aptamer, and a Raman-activemarker attached to the aptamer. The functionalized aptamer is covalentlyor non-covalently attached to the SERS-active surface. When this complexis contacted with a sample containing the aptamer binding target (i.e.,the analyte), the Raman-active marker on the aptamer is brought intoproximity of the SERS-active substrate and produces a strong Ramansignal with a signature spectrum characteristic of the Raman-activemarker. With the use of a different Raman-active marker for each aptamer(or aptamer set for a specific target), this assay can readily beadapted for multiplexing.

SUMMARY OF THE INVENTION

One embodiment of the invention is directed to a method of detecting atarget molecule in a sample which comprises contacting said sample withone or more target-specific aptamers covalently or non-covalentlyattached to a SERS-active surface, said one or more aptamers (each)having a Raman-active marker covalently attached thereto, such that uponbinding of said target to said one or more aptamers, said Raman-activemarker is in sufficient proximity of the Raman-active surface togenerate a detectable Raman signal. Hence, target-specific signal isdetected upon complex formation because the Raman active marker has beenbrought into proximity of the SERS-active substrate. This detectionscheme has many advantages over those known in the art. For example, itavoids the complexity of sandwich assays such as those used in ELISAformat and it looks for an increase in signal rather than signaldecrease often used in other types of assays.

The invention also provides target-specific aptamers with a covalentlybound Raman-active marker at the 5′ or 3′ terminus of the aptamer, oralternatively within 1-10 bases of the terminus of the aptamer, oralternatively anywhere along the aptamer backbone, so long as binding ofthe analyte induces the aptamer to undergo a conformation change, tothereby bring the Raman-active marker into close proximity with theSERS-active surface. The aptamer can be attached via a thiol linkage, orby other convenient moiety, the marker can be incorporated via amodified oligonucleotide or other covalent or non-covalent linkage. Inother embodiments, one or more oligonucleotides may be attached to theSERS active surface, which can be hybridized to the aptamer. In someembodiments, the aptamers of the invention are target-specific with aterminal thiol modification. In some embodiments, the Raman-activemarker is covalently conjugated to the aptamers at predeterminedlocations along the aptamer backbone. In some embodiments, the marker isFAM, TAMRA, Cy3, Texas-Red (TR), Cy3.5, Rhodamine 6G, Cy5, or the like,or combination thereof.

Another aspect of the invention provides a composition of mattercomprising a SERS-active surface covalently or coordinate covalentlybound with a target-specific DNA aptamer having a Raman-active markerintegrated into the DNA backbone at predetermined locations along itslength, and, on binding of said aptamer to its target, said marker beingsufficiently proximate to said SERS-active surface to produce a Ramansignature spectrum for said marker upon excitation.

Another aspect of the invention relates to a surface enhanced Ramanspectroscopy (SERS)-active reagent for detecting an analyte comprising:(a) one or more SERS-active surface; (b) unmodified or modified with oneor more aptamer; and (c) one or more Raman-active marker. In someembodiments, the reagent comprises in (c) unmodified or modified withone or more Raman-active marker.

In certain embodiments, the SERS-active surface is selected from thegroup consisting of metals (including but not limited to silver, gold,Cu, certain other transition metals and titanium nitride) semiconductorsubstrates (including but not limited to titanium oxide, zinc oxide,zinc selenide) or semimetals (including but not limited to graphene andmolybdenum disulfide). In certain embodiments, the SERS active surfacemay be a nanoparticle (NP) that is introduced into the biologicalsamples, or the SERS active material may be a solid support into whichNPs (SERS-active or inert) have or have not been embedded. The supportmaterial could be composed of materials including but not limited to:paper, cellulose, plastics including polystyrene, polyethylene andpolydimethyl siloxane (PDMS). In some embodiments, these supportmaterials are coated with one or several SERS-active materials. Anotherembodiment would be a patterned surface composed of one or several ofthose support materials coated with one or several SERS-activematerials. In certain embodiments, the aptamer is functionalized.

In certain embodiments, the aptamer is functionalized 1) with thiol tobind to the SERS-active surface and 2) with Raman-active markers toenhance detection.

In certain embodiments, the aptamer is covalently or non-covalentlyattached to the SERS-active surface.

In certain embodiments, the SERS-active surface is unmodified with oneor more aptamer.

In certain embodiments, the aptamer comprises a nucleotide sequenceselected from the group consisting of SEQ ID NOs: 1, 2, 3, 4, 5, 6, 7,8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25,26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43,44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61,62, 63, 64, 65, and 66.

In certain embodiments, the Raman-active marker comprises a dye orfluorescent marker.

In certain embodiments, the Raman-active marker is a fluorescent marker,and said fluorescent marker is selected from the group consisting offluorescein (FAM), Carboxytetramethylrhodamine (TAMRA), Cy3, Texas-Red(TR), Cy3.5, Rhodamine 6G, Cy5, TRIT (tetramethyl rhodamine isothiol),NBD (7-nitrobenz-2-oxa-1,3-diazole), phthalic acid, terephthalic acid,isophthalic acid, cresyl fast violet, cresyl blue violet, brilliantcresyl blue, para-aminobenzoic acid, erythrosine, biotin, digoxigenin,5-carboxy-4′,5′-dichloro-2′,7′-dimethoxy fluorescein,5-carboxy-2′,4′,5′,7′-tetrachlorofluorescein, 5-carboxyfluorescein,5-carboxy rhodamine, 6-carboxyrhodamine, 6-carboxytetramethyl aminophthalocyanines, azomethines, cyanines, xanthines, succinylfluoresceins,aminoacridine, quantum dots, carbon nanotubes, and fullerenes.

In certain embodiments, the Raman-active marker undergoes aconformational change upon binding of the analyte.

In certain embodiments, the aptamer undergoes a conformational changeupon binding of the analyte.

In certain embodiments, the conformational changes of the aptamer uponbinding of the analyte bring the Raman-active marker into closeproximity to the surface of the SERS-active surface and leads to anenhancement in the Raman signal.

In certain embodiments, the Raman-active marker is covalently attachedto the aptamer.

In certain embodiments, the analyte is selected from the groupconsisting of amino acid, peptide, polypeptide, protein, glycoprotein,lipoprotein, nucleoside, nucleotide, oligonucleotide, nucleic acid,sugar, carbohydrate, oligosaccharide, polysaccharide, fatty acid, lipid,hormone, metabolite, cytokine, chemokine, receptor, neurotransmitter,antigen, allergen, antibody, substrate, metabolite, cofactor, inhibitor,drug, pharmaceutical, nutrient, prion, toxin, poison, explosive,pesticide, chemical warfare agent, biohazardous agent, radioisotope,vitamin, heterocyclic aromatic compound, carcinogen, mutagen, narcotic,amphetamine, barbiturate, hallucinogen, waste product, drugs of abuse,contaminant and gunshot residue.

Yet another aspect of the invention relates to a diagnostic kitcomprising: a) at least one SERS-active reagent; said reagentcomprising: (i) one or more SERS-active surface; (ii) unmodified ormodified with one or more aptamer; and (iii) one or more Raman-activemarker; b) a Raman-active marker control to show that the Raman-baseddetection system is working; c) at least one blood, serum, other fluidor tissues sample, or laboratory prepared positive control; and c) atleast one blood, serum other fluid or tissues sample, or laboratoryprepared negative control. In some embodiments, the reagent comprises in(iii) unmodified or modified with one or more Raman-active marker.

In certain embodiments, the kit may further comprise a samplingcartridge.

Another aspect of the invention relates to a detection systemcomprising; a) unmodified or modified with one or more SERS-activereagent; said reagent comprising: (i) one or more SERS-active surface;(ii) one or more aptamer; and (iii) one or more Raman-active marker; andb) a Raman detector or Raman instrument. In some embodiments, thereagent comprises in (iii) unmodified or modified with one or moreRaman-active marker.

In certain embodiments, the Raman detector is portable.

In certain embodiments, the system further comprises a sample collectionapparatus.

Another aspect of the invention relates to a method for determining thepresence of one or more analyte in a biological sample, the methodcomprising: a) receiving a biological sample;

b) contacting the biological sample to at least one SERS-active reagentcomprising: (i) one or more SERS-active surface; (ii) unmodified ormodified with one or more aptamer; and (iii) one or more Raman-activemarker; c) allowing the analyte to come into contact with the aptamer;d) binding of the analyte by the aptamer, wherein said binding causesthe aptamer to undergo a conformational change; e) irradiating the atleast one SERS-active reagent bound to the one or more analyte; f)detecting the Raman signal to generate a Raman spectra; and g) comparingthe Raman signal detected in (f) with a reference Raman signal of acontrol, wherein the presence of one or more analyte in the biologicalsample is determined when said Raman signal detected in (f) differs fromsaid reference Raman signal. In certain embodiments of step (e), theSERS-active surface is bound to the aptamer, which recognizes theanalyte. In certain embodiments of step (g), the presence of one or moreanalyte in the biological sample is determined when said Raman signaldetected in (f) differs from the Raman signal in the absence of thetarget analyte. In some embodiments, the reagent comprises in (iii)unmodified or modified with one or more Raman-active marker.

Analogously, the invention relates to a method for determining thepresence of one or more analyte in a biological sample, the methodcomprising: a) obtaining a biological sample; b) contacting thebiological sample to at least one SERS-active reagent comprising: (i)unmodified or modified with one or more SERS-active surface; (ii) one ormore aptamer; and (iii) one or more Raman-active marker; c) allowing theanalyte to come into contact with the aptamer; d) binding of the analyteby the aptamer, wherein said binding causes the aptamer to undergo aconformational change; e) irradiating the at least one SERS-activereagent bound to the one or more analyte; f) detecting the Raman signalto generate a Raman spectra; and g) comparing the Raman signal detectedin (f) with a reference Raman signal of a control, wherein the presenceof one or more analyte in the biological sample is determined when saidRaman signal detected in (f) differs from said reference Raman signal.In some embodiments, the reagent comprises in (iii) unmodified ormodified with one or more Raman-active marker.

In certain embodiments, an increase in the Raman signal in the Ramanspectra in (f) compared to control is correlated with the amount of theone or more analyte.

In certain embodiments, the conformational change of the aptamer uponbinding of the analyte brings the Raman-active marker into closeproximity to the surface of the SERS-active material.

In certain embodiments, the method may be used to detect 1, 2, 3, 4, 5,6, 7, 8, 9, 10, or more analytes in a biological sample.

Another aspect of the invention relates to a method for diagnosing adisease or disorder in a subject comprising the steps of: a) receiving abiological sample from a subject; b) contacting the biological sample toat least one SERS-active reagent comprising: (i) one or more SERS-activesurface; (ii) unmodified or modified with one or more aptamer; and (iii)one or more Raman-active marker; c) allowing binding of the analyte byat least one SERS reagent in the biological sample, wherein said bindingcauses a conformational change to the one or more aptamer(s) of the SERSreagent; d) irradiating the at least one SERS-active reagent bound tothe one or more analyte; e) detecting the Raman signal of the at leastone SERS-active reagent; and f) comparing the Raman signal of said atleast one SERS-active reagent detected in (e) with a reference Ramansignal of said at least one SERS-active reagent detected in a biologicalsample received from a control subject (healthy subject), wherein saiddisease is diagnosed when said Raman signal detected in (e) differs fromsaid reference Raman signal in position and/or intensity of theRaman-sensitive marker peak. In some embodiments, the reagent comprisesin (iii) unmodified or modified with one or more Raman-active marker.

Analogously, the invention relates to a method for diagnosing a diseaseor disorder in a subject comprising the steps of: a) obtaining abiological sample from a subject; b) contacting the biological sample toat least one SERS-active reagent comprising: (i) one or more SERS-activesurface; (ii) unmodified or modified with one or more aptamer; and (iii)one or more Raman-active marker; c) allowing binding of the one or moreanalyte by at least one SERS reagent in the biological sample, whereinsaid binding causes a conformational change to the one or more aptamerof the SERS reagent; d) irradiating the at least one SERS-active reagentbound to the one or more analyte; e) detecting the Raman signal of theat least one SERS-active reagent; and f) comparing the Raman signal ofsaid at least one SERS-active reagent detected in (e) with a referenceRaman signal of said at least one SERS-active reagent detected in abiological sample received from a control subject (healthy subject),wherein said disease is diagnosed when said Raman signal detected in (e)differs from said reference Raman signal in position and/or intensity ofthe Raman-sensitive marker peak. In some embodiments, the reagentcomprises in (iii) unmodified or modified with one or more Raman-activemarker.

Alternatively, the invention relates to a method for diagnosing adisease or disorder in a subject, comprising: a) receiving a Ramansignal measured in a biological sample of a subject; b) receiving areference Raman signal measured in a biological sample of a controlsubject; and c) comparing the Raman signal of (a) with the referenceRaman signal of (b), wherein said disease is diagnosed when said Ramansignal in (a) differs in location and/or intensity from said referenceRaman signal in (b).

In certain embodiments, the disease or disorder is selected from thegroup consisting of an infectious disease, proliferative disease,neurodegenerative disease, cancer, psychological disease, metabolicdisease, autoimmune disease, sexually transmitted disease,gastro-intestinal disease, pulmonary disease, cardiovascular disease,stress- and fatigue-related disorder, fungal disease, pathogenicdisease, and obesity-related disorder.

The present invention is directed to reagents for and methods ofdetecting analytes bound by functionalized aptamers usingsurface-enhanced Raman scattering (SERS). The system is composed ofthree parts: a SERS-active surface or nanoparticle (henceforth referredto as SERS-active surface), an aptamer, and a Raman-active markerattached to the aptamer. The functionalized aptamer is covalently ornon-covalently attached to the SERS-active surface. When this complex iscontacted with a sample containing the aptamer binding target (i.e., theanalyte), the Raman-active marker on the aptamer is brought intoproximity of the SERS-active substrate and resulting in the observationof a strong Raman signal with a signature characteristic(s) of theRaman-active marker. With the use of a different Raman-active marker foreach aptamer (or aptamer set for a specific target), this assay canreadily be adapted for multiplexing.

Another aspect of the invention relates to a surface enhanced Ramanspectroscopy (SERS)-active reagent for detecting one or more analyte ina species of Borrelia comprising: (a) one or more SERS-active surface;(b) one or more aptamer directed to one or more analyte found in aspecies of Borrelia; and (c) one or more Raman-active marker.

In certain embodiments, the SERS-active surface is selected from thegroup consisting of metals (including but not limited to silver, gold,Cu, certain other transition metals and titanium nitride) semiconductorsubstrates (including but not limited to titanium oxide, zinc oxide,zinc selenide) or semimetals (including but not limited to graphene andmolybdenum disulfide).

In certain embodiments, the aptamer is functionalized.

In certain embodiments, the aptamer is functionalized 1) to bind theaptamer to the SERS-active surface and 2) with Raman-active markers toenhance detection.

In certain embodiments, the aptamer is covalently or non-covalentlyattached to the SERS-active surface.

In certain embodiments, the aptamer comprises a nucleotide sequencewhich is at least 80% identical to the nucleotide sequences selectedfrom the group consisting of SEQ ID NOs: 67-84.

In certain embodiments, the Raman-active marker comprises a dye, azide,alkyne, quantum dot, carbon nanotube, or fluorescent marker.

In certain embodiments, the Raman-active marker is a fluorescent marker,and said fluorescent marker is selected from the group consisting ofazides, alkynes, fluorescein (FAM), Carboxytetramethylrhodamine (TAMRA),Cy3, Texas-Red (TR), Cy3.5, Rhodamine 6G, Cy5, TRIT (tetramethylrhodamine isothiol), NBD (7-nitrobenz-2-oxa-1,3-diazole), phthalic acid,terephthalic acid, isophthalic acid, cresyl fast violet, cresyl blueviolet, brilliant cresyl blue, para-aminobenzoic acid, erythrosine,biotin, digoxigenin, 5-carboxy-4′,5′-dichloro-2′,7′-dimethoxyfluorescein, 5-carboxy-2′,4′,5′,7′-tetrachlorofluorescein,5-carboxyfluorescein, 5-carboxy rhodamine, 6-carboxyrhodamine,6-carboxytetramethyl amino phthalocyanines, azomethines, cyanines,xanthines, succinylfluoresceins, aminoacridine, quantum dots, carbonnanotubes, and fullerenes.

In certain embodiments, the aptamer conjugated to the Raman-activemarker undergoes reorganization upon binding of the analyte.

In certain embodiments, the aptamer conjugated to the Raman-activemarker undergoes a conformational change upon binding of the analyte.

In certain embodiments, the conformational change of the aptamer uponbinding of the analyte brings the Raman-active marker into closeproximity to the surface of the SERS-active surface and causes anenhancement in the Raman signal.

In certain embodiments, the Raman-active marker is covalently attachedto the aptamer.

In certain embodiments, the analyte is selected from the groupconsisting of surface protein, amino acid, peptide, polypeptide,protein, glycoprotein, lipoprotein, nucleoside, nucleotide,oligonucleotide, nucleic acid, sugar, carbohydrate, oligosaccharide,polysaccharide, fatty acid, lipid, hormone, metabolite, cytokine,chemokine, receptor, neurotransmitter, antigen, allergen, antibody,substrate, metabolite, cofactor, inhibitor, drug, pharmaceutical,nutrient, prion, toxin, poison, explosive, pesticide, chemical warfareagent, biohazardous agent, radioisotope, vitamin, heterocyclic aromaticcompound, carcinogen, mutagen, narcotic, amphetamine, barbiturate,hallucinogen, waste product, drugs of abuse, contaminant, and gun shotresidue.

In certain embodiments, the species of Borrelia is selected fromBorrelia afzelii, Borrelia americana, Borrelia andersonii, Borreliaanserina, Borrelia baltazardii, Borrelia bavariensis, Borreliabissettii, Borrelia brasiliensis, Borrelia burgdorferi, Borreliacaliforniensis, Borrelia carolinensis, Borrelia caucasica, Borreliacoriaceae, Borrelia crocidurae, Borrelia dugesii, Borrelia duttonii,Borrelia garinii, Borrelia graingeri, Borrelia harveyi, Borreliahermsii, Borrelia hispanica, Borrelia japonica, Borrelia kurtenbachii,Borrelia latyschewii, Borrelia lonestari, Borrelia lusitaniae, Borreliamazzottii, Borrelia merionesi, Borrelia microti, Borrelia miyamotoi,Borrelia parkeri, Borrelia persica, Borrelia recurrentis, Borreliasinica, Borrelia spielmanii, Borrelia tanukii, Borrelia texasensis,Borrelia theileri, Borrelia tillae, Borrelia turcica, Borrelia turdi,Borrelia turicatae, Borrelia valaisiana, Borrelia venezuelensis,Borrelia vincentii, Borrelia burgdorferi B31, Borrelia burgdorferi N40,Borrelia burgdorferi JD1, or Borrelia burgdorferi 297.

Another aspect of the invention relates to a diagnostic kit comprising:a) at least one SERS-active reagent; said reagent comprising: (i) one ormore SERS-active surface; (ii) one or more aptamer directed to one ormore analyte found in a species of Borrelia; and (iii) one or moreRaman-active marker; b) at least one positive control; and c) at leastone negative control.

Another aspect of the invention relates to a detection systemcomprising: a) one or more SERS-active reagent; said reagent comprising:(i) one or more SERS-active surface; (ii) one or more aptamer directedto one or more analyte found in a species of Borrelia; and (iii) one ormore Raman-active marker; and b) a Raman detector.

In certain embodiments, the Raman detector is portable or not portable.

In certain embodiments, the system further comprises a sample collectionapparatus.

Another aspect of the invention relates to a method for determining thepresence of one or more analyte in a biological sample, the methodcomprising: a) receiving a biological sample;

b) contacting the biological sample to at least one SERS-active reagentcomprising: (i) one or more SERS-active surface; (ii) one or moreaptamer directed to one or more analyte found in a species of Borrelia;and (iii) one or more Raman-active marker; c) allowing the analyte tocome into contact with the aptamer; d) binding of the analyte by theaptamer, wherein said binding causes the one or more aptamer(s) toundergo a conformational change; e) irradiating the at least oneSERS-active reagent bound to the one or more aptamer; f) detecting theRaman signal to generate a Raman spectrum; and g) comparing the Ramansignal detected in (f) with a reference Raman signal of a control,wherein the presence of one or more analyte in the biological sample isdetermined when said Raman signal detected in (f) differs from saidreference Raman signal in position and/or intensity of the peakassociated with Raman-sensitive marker. The methods of the presentinvention allows for the direct detection of Borrelia and/or thedetection of anti-Borrelia antibodies.

Analogously, the invention relates to a method for determining thepresence of one or more analyte in a biological sample, the methodcomprising: a) obtaining a biological sample; b) contacting thebiological sample to at least one SERS-active reagent comprising: (i)one or more SERS-active surface; (ii) one or more aptamer directed toone or more analyte found in a species of Borrelia; and (iii) one ormore Raman-active marker; c) allowing the analyte to come into contactwith the aptamer; d) binding of the analyte by the aptamer, wherein saidbinding causes the one or more aptamer to undergo a conformationalchange; e) irradiating the at least one SERS-active reagent bound to theone or more analyte; f) detecting the Raman signal to generate a Ramanspectra; and g) comparing the Raman signal detected in (f) with areference Raman signal of a control, wherein the presence of one or moreanalyte in the biological sample is determined when said Raman signaldetected in (f) differs from said reference Raman signal. The methods ofthe present invention allows for the direct detection of Borrelia, asopposed to the detection of anti-Borrelia antibodies.

In certain embodiments, an increase in the Raman signal in the Ramanspectra in (f) compared to control is correlated with the amount of theone or more analyte.

In certain embodiments, the conformational change of the aptamer uponbinding of the analyte brings the Raman-active marker into closeproximity to the surface of the SERS-active nanoparticle.

In certain embodiments, the method may be used to detect 1, 2, 3, 4, 5,6, 7, 8, 9, 10, or more analytes in a biological sample.

Another aspect of the invention relates to a method for diagnosing aLyme disease in a subject comprising the steps of: a) receiving abiological sample from a subject; b) contacting the biological sample toat least one SERS-active reagent comprising: (i) one or more SERS-activesurface(s); (ii) one or more aptamer directed to one or more analytefound in a species of Borrelia; and (iii) one or more Raman-activemarker; c) allowing binding of the at least one SERS reagent by one ormore analyte in the biological sample, wherein said binding causes aconformational change to the one or more aptamers of the SERS reagent;d) irradiating the at least one SERS-active reagent bound to the one ormore analyte; e) detecting the Raman signal of the at least oneSERS-active reagent; and f) comparing the Raman signal of said at leastone SERS-active reagent detected in (e) with a reference Raman signal ofsaid at least one SERS-active reagent detected in a biological samplereceived from a control subject (healthy subject), wherein said Lymedisease is diagnosed when said Raman signal detected in (e) differs fromsaid reference Raman signal. In certain embodiments of step (d), theSERS-active reagent is bound to the aptamer, which recognizes theanalyte. In certain embodiments of step (f), said Raman signal detectedin (e) differs from the Raman signal in the absence of the targetanalyte.

Analogously, the invention relates to a method for diagnosing a Lymedisease in a subject comprising the steps of: a) obtaining a biologicalsample from a subject; b) contacting the biological sample to at leastone SERS-active reagent comprising: (i) one or more SERS-active surface;(ii) one or more aptamer directed to one or more analyte found in aspecies of Borrelia; and (iii) one or more Raman-active marker; c)allowing binding of the at least one SERS reagent by one or more analytein the biological sample, wherein said binding causes a conformationalchange to the one or more aptamers of the SERS reagent; d) irradiatingthe at least one SERS-active reagent bound to the one or more aptamer;e) detecting the Raman signal of the at least one SERS-active reagent;and f) comparing the Raman signal of said at least one SERS-activereagent detected in (e) with a reference Raman signal of said at leastone SERS-active reagent detected in a biological sample received from anegative control subject (healthy subject), pooled samples from numerousnegative control subjects, or a laboratory prepared negative controlsample wherein said Lyme disease is diagnosed when said Raman signaldetected in (e) differs from said reference Raman signal.

Alternatively, the invention relates to a method for diagnosing Lymedisease in a subject, comprising: a) receiving a Raman signal measuredin a biological sample of a subject; b) receiving a reference Ramansignal measured in a biological sample of a control subject; and c)comparing the Raman signal of (a) with the reference Raman signal of(b), wherein said Lyme disease is diagnosed when said Raman signal in(a) differs in position and/or intensity from said reference Ramansignal in (b).

Other objects, features and advantages of the present invention willbecome apparent from the following detailed description. It should beunderstood, however, that the detailed description and the specificexamples, while indicating preferred embodiments of the invention, aregiven by way of illustration only, since various changes andmodifications within the spirit and scope of the invention will becomeapparent to those skilled in the art from this detailed description.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 depicts a schematic figure showing the in vitro evolution ofaptamers using SELEX. Typically 8-12 rounds of selection are used togenerate tight binding, high specificity aptamers. (Figure from:http://sitemaker.umich.edu/ntr/aptamers)

FIG. 2 contains six panels. Panel A depicts a comparison of absorptionspectra of silver NPs produced using the microwave-based syntheticmethod. The plasmon absorption maximum at 401 nm, and a FWHM of 60 nmare maintained by the Ag NPs over a three week period. Shown are theabsorption spectra of NPs on the day they were synthesized (blue), andthe same NPs three weeks after synthesis (red), and NPs stored in thereaction mixture which were centrifuged three weeks after synthesis(green), demonstrating the ability of the NPs to maintain the observedspectral characteristics was not dependent on the method ofpurification. Panel B depicts a comparison of Raman spectra of 200 μMsolutions of mercaptophenol (MCP) in the presence (blue) and absence(red) of Ag NPs. The asterisk indicates the spectral contribution of thesilicon substrate on which the sample was dried. Panel C depictsabsorption spectra of dithiol terminated DNA-modified Ag NPs uponaddition of NaCl. Addition of 25 mM NaCl resulted in a decrease inabsorption intensity that achieved a steady absorption after 15 min.Increasing the NaCl concentration to 50 mM resulted in further decreasein the intensity of the plasmon absorption. Addition of a third aliquotresulted in nearly complete loss of the plasmon absorption immediatelyafter addition of the aliquot. Panels D and E depict spectralinterrogation of the surface modification of Ag NPs withdithiol-modified DNA. Panel D shows a representative spectral analysisof DNA-modified Ag NP plasmon absorption of NPs modified with DNA in theabsence of a reducing agent. The spectra were recorded to follow theeffect on the plasmon absorption of the addition 0 to 100 mM sodiumchloride in 25 mM increments. Panel E shows a comparison of decrease inplasmon absorption intensity with addition of sodium chloride to Ag NPsmodified with dithiol-terminated DNA. The change in absorption of theplasmon at 403 nm is shown as a fraction of the initial plasmonabsorption intensity. Spectra were collected until the plasmonabsorption intensity dropped below 50% of the initial intensity. Panel Fdepicts emission spectra of 5′-FAM, 3′-dithiol modified DNA after DTTtreatment. FAM emission spectra resulting from DTT treatment of Ag NPstreated with DNA in the presence of TCEP reduced—(red, bottom) anddisulfide DNA in the absence of reducing agent (blue, top).

FIG. 3 contains six panels. Panel A depicts comparison of Raman spectraof 200 μM aqueous solutions of mercaptophenol (MCP) in the presence of100 μM solutions of potassium nitrate (red, top), or sodium chloride(green), and in the absence of salt (blue, bottom). Panel B depictscomparison of the efficacy of salt additives on increasing Raman signalintensity. Raman spectra of AgNP surface modified with 5′-FAMdithiolated DNA reveal the addition of KNO₃ (green) produce more intensespectral features than in the presence of NaCl (red) or in the absenceof added salt (blue) in producing the most intense spectral features.The DNA modification was performed in the presence of 50 mM NaCl. PanelC depicts a comparison of Raman spectra of Ag NPs modified with mixedmonolayer composed of mercaptophenol and FAM-terminated DNA. Spectrashown, from bottom to top, are of Ag NPs with a monolayer of 4 μMmercaptophenol and mixed monolayers of FAM-modified DNA withconcentrations of MCP ranging from 4 mM to 4 μM decreasing by an orderof magnitude in each spectrum. Panel D depicts a comparison of Ramanspectra of Ag NPs modified with mercaptophenol (MCP; bottom),FAM-terminated DNA (middle) and a mixed monolayer of FAM-terminated DNAwith 4 mM MCP (top). All spectra offset for clarity; data collected with632.8 nm incident light. The color of the arrow matches the source ofthe spectral feature; red arrows indicate contributions from MCP, andblack arrows indicate contributions from FAM-terminated DNA. Panel Edepicts a comparison of Raman spectra of Ag NPs modified withmercaptophenol (MCP; bottom), FAM-terminated DNA (middle) and a mixedmonolayer of FAM-terminated DNA with 4 mM MCP (top) collected with 785nm incident light. Arrows indicate spectral features derived from thecomponents of the mixed monolayer. The features at 305 cm⁻¹, 714 cm⁻¹and 1050 cm⁻¹ are derived from the FAM dye found at the 5′-terminus ofthe dithiol-modified DNA, whereas the features at 393 cm⁻¹, 636 cm⁻¹,1010 cm⁻¹, 1080 cm⁻¹ and 1175 cm⁻¹ are contributions from the MCPmonolayer. Panel F depicts a comparison of effect of concentration ofMCP and MCH on spectral intensities of MCP and FAM on mixed monolayer AgNPs. Shown are the spectral intensities observed of the FAM-derivedfeature (1050 cm⁻¹; blue and green) and MCP derived feature (1076 cm⁻¹;red and purple) at two concentrations of MCP. The spectral intensity ofthe two components of the mixed monolayer collected with 40 μM MCP (blueand red), and 4 μM MCP (green and purple).

FIG. 4 contains two panels. Panel A depicts Ag NPs modified withTAMRA-containing vasopressin-targeted aptamers demonstrate selectivityfor vasopressin against structurally related proteins: oxytocin andsubstance P. A linear correlation was observed with vasopressin (R²:0.99), whereas the interaction between substance P (R²: 0.63) andoxytocin (R²: 0.19) did not result in a linear correlation betweenconcentration and spectral intensity. Panel B depicts a comparison ofmonolayer compositions of Ag NPs modified with TAMRA-containingvasopressin-targeted aptamers. Modification of the Ag NP surface witheither the vasopressin-targeted aptamer alone, or in tandem with 40 μMMCP (R²: 0.99 and 0.96, respectively), resulted in a linear correlationbetween vasopressin concentration and spectral feature intensity. Incontrast, NPs modified with vasopressin-targeting aptamer and 4 M MCHdid not result in a linear correlation (R²: 0.03).

FIG. 5 contains two panels. Panel A depicts absorption spectrum ofsilver NPs produced using the microwave-based synthetic methods in whichthe solution was heated to 120° C. over 30 seconds, and held at thistemperature for 30, 60 and 90 seconds. The NPs produced exhibit spectralcharacteristics (401 nm; FWHM=60 nm) that compare favorably with spectrafrom literature. Panel B depicts an example absorption spectrum ofmicrowave synthesized Ag NPs from Leona, M. Proc. Natl. Acad. Sci. 2009,106, 14757.

FIG. 6 contains two panels. Panel A depicts a schematic representationof the SERS-based detection of Borrelia burgdorferi from human blood.Drops of blood, serum, or plasma are used directly for the detection ofB. burgdorferi-specific proteins. Blood, serum, or plasma is exposed toaptamer coated SERS-active surface, filtered, and interrogated by Ramanspectroscopy. Spectra are analyzed on a small instrument and results areavailable in <30 min. Panel B depicts a schematic representation of theproposed SERS-based detection of Borrelia burgdorferi in Ixodesscapularis nymphs. Blacklegged tick nymphs are collected and lysed. Thehomogenate is mixed with aptamer coated nanoparticles, and interrogatedby Raman spectroscopy. Spectra are analyzed on the portable instrumentand results are available in <20 min.

FIG. 7 contains two panels depicting aptamer selection workflow formultiple targets by use of micro-columns. Panel A shows micro-columnfilled with 10 μL of GFP-immobilized resin. Panel B shows multiplexedselection of RNA aptamers. The steps shown with dashed arrows areoptional and are not necessarily done in each round. (Szeto, K. et al.PLoS ONE 2013, 8, e82667).

FIG. 8 depicts high affinity binding of OspA aptamer (SEQ ID NO: 4) toB. burgdorferi OspA protein. K_(d) for binding is approximately 2.2 nMas determined by fluorescence anisotropy.

FIG. 9 contains two panels depicting the schematic representation (PanelA) and demonstration of the principle of Raman-active marker modifiedaptamers for detection of biological targets (Panel B). Panel A showsthe Raman active dye (represented by blue circle) is brought in closeproximity to the Raman substrate surface (represented by yellow block)upon exposure to the target analyte (represented by red circle). Theclose proximity of the dye and the SERS substrate result in highintensity SERS signals facilitating detection of the four targetanalytes (figure derived from: Baker, B. R. et al. J. Am. Chem. Soc.2006, 128, 3138). Panel B depicts spectra demonstrating theprinciple—the present inventors modified a vasopressin-targeting aptamerwith TAMRA, a Raman active dye. The intensity of the TAMRA Raman signalwas observed to increase with concentration of the target moleculevasopressin, demonstrating the efficacy of Raman-active dye-modifiedaptamers in detection of biological targets.

FIG. 10 depicts Raman spectra of Raman-active dyes to be employed in themulti-plex detection. Each dye serves as a proxy for each of the fourtarget analytes, has spectral features that allow their identificationin the spectra of samples containing the other dyes, and facilitateanalyte detection in saliva or other biological matrices. (Cao, Y. C. etal. Science 2002, 297, 1536).

DETAILED DESCRIPTION A. Definitions

For convenience, certain terms employed in the specification, examples,and appended claims are collected here. Unless defined otherwise, alltechnical and scientific terms used herein have the same meaning ascommonly understood by one of ordinary skill in the art to which thisinvention belongs.

The articles “a” and “an” are used herein to refer to one or to morethan one (i.e., to at least one) of the grammatical object of thearticle. By way of example, “an element” means one element or more thanone element.

B. Nanoparticles and Other SERS Active Substrate Materials

SERS-active nanoparticles are used to provide the Raman-active surfaceof the invention and are known in the art. Other SERS-activematerial-coated surfaces, metal-coated surfaces, or surfaces withembedded SERS-active nanoparticles are contemplated by the invention.SERS-active nanoparticles include, but are not limited to, thosedescribed in U.S. Patent Appln. Pub. No. 2004/0134997. For example,silver (Ag) nanoparticles can be prepared using a microwave-basedreduction of AgNO₃ or by any number of other methods known to those ofskill in the art. Silver and gold nanoparticles are also known and usedand can be obtained commercially or synthetically and adapted for use inthe present methods. In addition to nanoparticles other SERS activesubstrates can be used including, but not limited to: core shell, hollowor Si beads coated with metals for Raman activity, or other SERS activesubstrates composed of metals, semiconductors or, semi-metals onappropriate supports, or polymeric surfaces coated with SERS-activematerials; these materials may or may not have NPs embedded. Wang, W.;et al., Appl. Phys. Lett. 106, 20.15, 211604.

In certain embodiments, the SERS active NP may be free and introducedinto the biological samples, or the SERS active material may be a solidsupport into which NPs (SERS-active or inert) have been embedded. Thesupport material could be composed of materials including but notlimited to: paper, cellulose, plastics including polystyrene,polyethylene and polydimenthyl siloxane (PDMS) or other polymericmaterials. In some embodiments, these support materials are coated withone or several SERS-active materials. Another embodiment would be apatterned surface composed of one or several of those support materialscoated with one or several SERS-active materials (Wang, W.; et al.,Appl. Phys. Lett. 106, 2015, 211604)

If aptamers are attached to the SERS active substrate, aptamers can bebound can be bound to the SERS-active surface either covalently ornon-covalently. The DNA aptamer may be modified with the thiol. Forcovalent, dative or coordinate covalent attachment, the surface orSERS-active substrates can be modified to contain various reactivegroups suitable for attaching DNA or RNA aptamers or oligonucleotides,typically thiols but any groups known in the art can be used. Forexample, a capping reagent such as dihydrolipoic acid can be covalentlyattached to the Raman-active surface and amine-terminated DNA aptamercan be covalently linked to the nanoparticle using standard amine tocarboxylic acid conjugation with EDC(1-ethyl-3-(3-dimethylaminopropyl)carbodiimide). For non-covalentaptamer attachment, a barcode DNA, universal primer or other bindingspecific sequence of DNA (or RNA), i.e., a capture oligo, can beattached to the nanoparticles or SERS-active surfaces. Such captureoligos are hybridized with target-specific aptamers which have thecomplementary sequence of the capture oligo (typically at a significantdistance or near the opposite end from the Raman active marker).

C. Aptamers

Aptamers are single-stranded nucleic acid (DNA or RNA) molecules,typically but not always, under 100 bases in length, which have theability to bind to other molecules with high affinity and specificity.Aptamers can be generated, for example, using an in vitro evolutionaryprocess using random oligonucleotide pools by a process calledsystematic Evolution of Ligands by EXponential enrichment (SELEX; FIG.1). The SELEX process is controlled by the ability of these smalloligonucleotides to fold into unique three dimensional structures thatcan interact with a specific target with high specificity and affinity.Aptamers can be been generated against a wide variety of targets,including: metal ions,¹ small molecules such as organic dyes² and aminoacids,³ medically relevant molecules such as antibiotics⁴ and peptides,⁵and biologically relevant molecules such as proteins,^(6,7) whole cells,viruses and virus-infected cells,⁸ and bacteria.^(9,10) Once thesequences for a particular aptamer or aptamer set is known, the aptamerscan readily be synthesized using standard techniques known in the artfor oligonucelotides or via synthetic or recombinant DNA techniques.Aptamers that bind oxytocin, vasopressin, other hormones, infectiousdisease agents, and disease marker proteins are of particular interest.

In addition, the aptamers may bind illicit drugs, such as but notlimited to, cannabinoids/Cannabis/Marijuana (Δ9-Tetrahydrocannabinol,THC), synthetic cannabinoids, Carisoprodol (and Meprobamate), Cocaine(Methylbenzoylecgonine), Dextromethorphan, Diphenhydramine,Gamma-Hydroxybutyrate (GHB, GBL, and 1,4-BD), Ketamine, Lysergic aciddiethylamide (LSD), buprenorphine (subutex), Methadone, Methamphetamine,Amphetamine, Methylenedioxymethamphetamine (MDMA, Ecstasy),barbiturates, benzodiazepines, opiates (Oxycodone, propoxyphene,Morphine and Heroin), or Phencyclidine (PCP).

The aptamers of the present invention are useful for diagnostic andprognostic applications such as detecting persistent infectious disease,proliferative diseases, neurodegenerative diseases, cancers,psychological diseases, metabolic diseases, autoimmune diseases,sexually transmitted diseases, gastro-intestinal diseases, pulmonarydiseases, cardiovascular diseases, stress- and fatigue-relateddisorders, fungal diseases, pathogenic diseases, obesity-relateddisorders, or biomarkers regarding same. Viral infectious diseasesincluding human papilloma virus (HPV), hepatitis A Virus (HAV),hepatitis B Virus (HBV), hepatitis C Virus (HCV), retroviruses such ashuman immunodeficiency virus (HIV-1 and HIV-2), herpes viruses such asEpstein Barr Virus (EBV), cytomegalovirus (CMV), HSV-1 and HSV-2,influenza virus, Hepatitis A and B, FIV, lentiviruses, pestiviruses,West Nile Virus, measles, smallpox, cowpox, ebola, coronavirus,retrovirus, herpesvirus, potato S virus, simian Virus 40 (SV40), MouseMammary Tumor Virus (MMTV) promoter, Moloney virus, ALV, Cytomegalovirus(CMV), Epstein Barr Virus (EBV), or Rous Sarcoma Virus (RSV). Theaptamers of the present invention may detect antigens, antibodies, orother analytes associated with pathogens such as various parasites, likemalaria. In addition, bacterial, fungal and other pathogenic diseasesare included, such as Aspergillus, Brugia, Candida, Chikungunya,Chlamydia, Coccidia, Cryptococcus, Dengue, Dirofilaria, Gonococcus,Histoplasma, Leishmania, Mycobacterium, Mycoplasma, Paramecium,Pertussis, Plasmodium, Pneumococcus, Pneumocystis, P. vivax in Anophelesmosquito vectors, Rickettsia, Salmonella, Shigella, Staphylococcus,Streptococcus, Toxoplasma and Vibriocholerae. Exemplary species includeNeisseria gonorrhea, Mycobacterium tuberculosis, Candida albicans,Candida tropicalis, Trichomonas vaginalis, Haemophilus vaginalis, GroupB Streptococcus sp., Microplasma hominis, Hemophilus ducreyi, Granulomainguinale, Lymphopathia venereum, Treponema pallidum, Brucella abortus.Brucella melitensis, Brucella suis, Brucella canis, Campylobacter fetus,Campylobacter fetus intestinalis, Leptospira pomona, Listeriamonocytogenes, Brucella ovis, Chlamydia psittaci, Trichomonas foetus,Toxoplasma gondii, Escherichia coli, Actinobacillus equuli, Salmonellaabortus ovis, Salmonella abortus equi, Pseudomonas aeruginosa,Corynebacterium equi, Corynebacterium pyogenes, Actinobaccilus seminis,Mycoplasma bovigenitalium, Aspergillus fumigatus, Absidia ramosa,Trypanosoma equiperdum, Clostridium tetani, Clostridium botulinum; or, afungus, such as, e.g., Paracoccidioides brasiliensis; or other pathogen,e.g., Plasmodium falciparum. Also included are National Institute ofAllergy and Infectious Diseases (NIAID) priority pathogens. Theseinclude Category A agents, such as variola major (smallpox), Bacillusanthracis (anthrax), Yersinia pestis (plague), Clostridium botulinumtoxin (botulism), Clostridium difficile, Francisella tularensis(tularaemia), filoviruses (Ebola hemorrhagic fever, Marburg hemorrhagicfever), arenaviruses (Lassa (Lassa fever), Junin (Argentine hemorrhagicfever) and related viruses); Category B agents, such as Coxiellaburnetti (Q fever), Brucella species (brucellosis), Burkholderia mallei(glanders), alphaviruses (Venezuelan encephalomyelitis, eastern &western equine encephalomyelitis), ricin toxin from Ricinus communis(castor beans), epsilon toxin of Clostridium perfringens; Staphylococcusenterotoxin B, Salmonella species, Shigella dysenteriae, Escherichiacoli strain O157:H7, Vibrio cholerae, Cryptosporidium parvum; Category Cagents, such as nipah virus, hantaviruses, yellow fever in Aedesmosquitoes, and multidrug-resistant tuberculosis; helminths, such asSchistosoma and Taenia; and protozoa, such as Leishmania (e.g., L.mexicana) in sand flies, Plasmodium, Chagas disease in assassin bugs.

Bacterial pathogens include, but are not limited to, such as bacterialpathogenic gram-positive cocci, which include but are not limited to:pneumococci; staphylococci; and streptococci. Pathogenic gram-negativecocci include: meningococci; and gonococci. Pathogenic entericgram-negative bacilli include: enterobacteriaceae; Pseudomonas,acinetobacteria and eikenella; melioidosis; Salmonella; shigellosis;hemophilus; chancroid; brucellosis; tularemia; Yersinia (Pasteurella);Streptobacillus moniliformis and spirilum; Listeria monocytogenes;erysipelothrix rhusiopathiae; diphtheria; cholera; anthrax; anddonovanosis (Granuloma inguinale). Pathogenic anaerobic bacteriainclude; tetanus; botulism; other clostridia; tuberculosis; leprosy; andother mycobacteria. Pathogenic spirochetal diseases include: syphilis;treponematoses: yaws, pinta and endemic syphilis; and leptospirosis.Other infections caused by higher pathogen bacteria and pathogenic fungiinclude: actinomycosis; nocardiosis; cryptococcosis, blastomycosis,histoplasmosis and coccidioidomycosis; candidiasis, aspergillosis, andmucormycosis; sporotrichosis; paracoccidiodomycosis, petriellidiosis,torulopsosis, mycetoma and chromomycosis; and dermatophytosis.Rickettsial infections include rickettsial and rickettsioses. Examplesof mycoplasma and chlamydial infections include: Mycoplasma pneumoniae;lymphogranuloma venereum; psittacosis; and perinatal chlamydialinfections. Pathogenic protozoans and helminths and infectionseukaryotes thereby include: amebiasis; malaria; leishmaniasis;trypanosomiasis; toxoplasmosis; Pneumocystis carinii; giardiasis;trichinosis; filariasis; schistosomiasis; nematodes; trematodes orflukes; and cestode (tapeworm) infections.

The aptamers of the present invention are useful for diagnostic andprognostic applications such as detecting persistent infectious disease.In certain embodiments, the aptamers may detect infections caused byspecies in the family of Borrelia, such as B. burgdorferi infections, inhumans using human blood, serum or plasma, taken from a finger stick orstandard blood draw. In addition, the aptamers may detect Lyme and othertick-borne pathogens, such as: Babesia, Ehrlichia, Anaplasma,Bartonella, and other emerging tick borne pathogens such as Borreliamiyamotoi, spotted fevers, and Powassan virus. The aptamer may be usedto detect Borrelia afzelii, Borrelia americana, Borrelia andersonii,Borrelia anserina, Borrelia baltazardii, Borrelia bavariensis, Borreliabissettii, Borrelia brasiliensis, Borrelia burgdorferi, Borreliacaliforniensis, Borrelia carolinensis, Borrelia caucasica, Borreliacoriaceae, Borrelia crocidurae, Borrelia dugesii, Borrelia duttonii,Borrelia garinii, Borrelia graingeri, Borrelia harveyi, Borreliahermsii, Borrelia hispanica, Borrelia japonica, Borrelia kurtenbachii,Borrelia latyschewii, Borrelia lonestari, Borrelia lusitaniae, Borreliamazzottii, Borrelia merionesi, Borrelia microti, Borrelia miyamotoi,Borrelia parkeri, Borrelia persica, Borrelia recurrentis, Borreliasinica, Borrelia spielmanii, Borrelia tanukii, Borrelia texasensis,Borrelia theileri, Borrelia tillae, Borrelia turcica, Borrelia turdi,Borrelia turicatae, Borrelia valaisiana, Borrelia venezuelensis,Borrelia vincentii, Borrelia burgdorferi B31, Borrelia burgdorferi N40,Borrelia burgdorferi JD1, or Borrelia burgdorferi 297. The Borreliaspecies may be found in Ixodes scapularis ticks.

“Babesia” refers to infectious protozoan species of the Babesia family,including but not limited to, Babesia bigemina, Babesia bovis, Babesiacanis, Babesia cati, Babesia divergens, Babesia duncani, Babesia felis,Babesia gibsoni, Babesia herpailuri, Babesia jakimovi, Babesia major,Babesia microti, Babesia ovate, or Babesia pantherae. “Ehrlichia” refersto the infections pathogenic species of the Ehrlichia family, includingbut not limited to, Ehrlichia chaffeensis, Ehrlichia muris, Ehrlichiaewingii, Ehrlichia ruminantium, or Ehrlichia canis. “Anaplasma” refersto the infections pathogenic species of the Anaplasma family, includingbut not limited to, Anaplasma phagocytophilum, Anaplasma marginale,Anaplasma centrale, Anaplasma mesaeterum, Anaplasma ovis, or Anaplasmaplatys. “Bartonella” refers to the infections pathogenic species of theBartonella family, including but not limited to Bartonella henselae,Bartonella quintana, Bartonella bacilliformis, Bartonella elizabethae,or Bartonella clarridgeiae.

On the basis of their target-recognition capability, selectivity andhigh affinity binding, aptamers have been likened to antibodies.However, aptamers, by their unique features, have more flexibility intheir development and range of applications. Specifically, the timeneeded for the generation of aptamers by the SELEX process iscomparatively short. In addition, aptamers can be chemicallysynthesized, which permits the biochemical manipulation required toincorporate various functional groups and specific moieties such asbiotin, carboxyl, amino and thiol groups, most of which do not affectthe recognition of the target by the aptamer. Aptamers are amenable toin vitro evolution, where increased pressure can be applied during theselection process, potentially increasing the affinity or selectivity ofthe aptamers for their target.

For example, aptamers are selected from a pool of 10¹⁴ to 10¹⁵ randomDNA or RNA sequence aptamers (purchased as a pool from a commercialsource that produces synthetic oligonucleotides), and thesequence-specific aptamers are enriched using the SELEX process. SELEXinvolves exposure of the DNA or RNA aptamer pool to a target, typicallyon solid support, as well as “negative” targets (i.e., other parts ofthe process, such as the plastic used in the process or the solidsupport in the absence of the target) to ensure the pool is not enrichedfor “negative” targets. The aptamers interact with and are bound to thetarget, and non-bound aptamers are washed away. The bound aptamers areeluted and copied by PCR, using the flanking constant region asamplification primer sites. This process is repeated with the enrichedaptamer pool until a few aptamers become the majority of the pool. Gelshift assays, fluorescence anisotropy experiments or other bindingexperiments can be used to determine the affinity of the aptamers for agiven target.

The term “% homology” is used interchangeably herein with the term “%identity” herein and normally refers to the level of nucleic acididentity between the nucleic acid sequence of the DNA or RNA aptamers ofthe present inventions, when aligned using a sequence alignment program.

For example, as used herein, 80% homology means the same thing as 80%sequence identity determined by a defined algorithm, and accordingly ahomologue of a given sequence has greater than 80% sequence identityover a length of the given sequence. Exemplary levels of sequenceidentity include, but are not limited to, 80, 85, 90, 95, 98% or moresequence identity to a given sequence as described herein.

Exemplary computer programs which can be used to determine identitybetween two sequences include, but are not limited to, the suite ofBLAST programs, e.g., BLASTN, BLASTX, and TBLASTX, BLASTP and TBLASTN,publicly accessible at www.ncbi.nlm.nih.gov/BLAST, and other nextgeneration DNA sequencing analysis programs. Other programs includeGalaxy, Lasergene Genomics Suite, CLC Genomics Workbench, DNANexus,GenomeQuest, Softgene NextGENe

In certain embodiment, the present invention includes aptamerscomprising a nucleotide sequence that is at least 50% identical to anucleotide sequence selected from the group consisting of: SEQ ID Nos.1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20,21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38,39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56,57, 58, 59, 60, 61, 62, 63, 64, 65, and 66, or any combinations thereof.In certain embodiments, the aptamers comprise a nucleotide sequence thatis at least 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%,95%, 96%, 97%, 98%, 98.1%, 98.2%, 98.3%, 98.4%, 98.5%, 98.6%, 98.7%,98.8%, 98.9%, 99%, 99.1%, 99.2%, 99.3%, 99.4%, 99.5%, 99.6%, 99.7%,99.8%, or 99.9% identical to a nucleotide sequence selected from thegroup consisting of: SEQ ID Nos. 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12,13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30,31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48,49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, and66, or any combinations thereof.

Exemplary aptamer sequences for lyme detection are provided in Table 1.

TABLE 1 Aptamers directed to OspA, OspC, and BmpA OspA-21CATGACACCGTACCTGCTCTAATAAGCACGCCAGGGACTATTAGATCGGAATAGCACACGTCTGAACTCCAAGCACGCCAGGGACTATTA (SEQ ID NO: 67) OspA-46CATGACACCGTACCTGCTCTAATAAGCACGCCAGGGACTATTAGATCGGAAGAGCACACGTGTGAACTCCAAGCACGCCAGGGACTATTA (SEQ ID NO: 68) OspA-22CATGACACCGTACCTGCTCTACGAGATTCAAGCACTCCAGGGACTATTAGATCGGAAGAGCACACGTCTGAAGCACGCCAGGGACTATTA (SEQ ID NO: 69) OspA-39CATGACACCGTACCTGCTCTACGAGATTCAAGCACGCCAGGGATTATTAGATCGGAAGAGCACACGTCTGAAGCACGCCAGGGACTATTA (SEQ ID NO: 70) OspA-55CATGACACCGTACCTGCTCTTGCTTTTCGTGCGCGCATAAAATACTTTGATACTGTGCCGGATGAAAGCGAAGCACGCCAGGGACTATTA (SEQ ID NO: 71) OspA-59CATGACACCGTACCTGCTCTTGCTTTTCGTGCGCGCATAAAATACCTTGATACTGTGCCGTATGAAAGCGAAGCACGCCAGGGACTATTA (SEQ ID NO: 72) OspC-23CATGACACCGTACCTGCTCTGCGGTGCTGTATCGTCGTTTAGGCTGTTACCAGGGCCACCGGACAGAGGTAAGCACGCCAGGGACTATTA (SEQ ID NO: 73) OspC-28CATGACACCGTACCTGCTCTCGTATAGATCCTCTCGCGCTTCGGTTTTTAGAAGTATTCAAGGTATCATCAAGCACGCCAGGGACTATTA (SEQ ID NO: 74) OspC-30CATGACACCGTACCTGCTCTGATCAGCCTGGTCAACGGGTGGTCCTGTGCCAAGCTCGAAAATTCGCCGAAAGCACGCCAGGGACTATTA (SEQ ID NO: 75) OspC-34CATGACACCGTACCTGCTCTTGGAGCTAGAGAGCCGGTGATCGAAATTCTGGATGTTTCTGACGTTTGCTAAGCACGCCAGGGACTATTA (SEQ ID NO: 76) OspC-36CATGACACCGTACCTGCTCTACCCCGGAAATGATTAGCCATTGTGGTACTCATCTGGGCAGTCAGCACATAAGCACGCCAGGGACTATTA (SEQ ID NO: 77) OspC-37CATGACACCGTACCTGCTCTTTAACCCCTCGCGGAGGTGTACACGGGCCTACATAATCCTCCGAGGTTCCAAGCACGCCAGGGACTATTA (SEQ ID NO: 78) BmpA-5CATGACACCGTACCTGCTCTTTACGTTTGGGACGTCTGGCGAAGCCACCACAAGCTAGCCCTCCAATTTAAAGCACGCCAGGGACTATTA (SEQ ID NO: 79) BmpA-6CATGACACCGTACCTGCTCTTTGATCATCACGGCACACTCATTACGGTTGGATATACTAGTCCGGTTAGAAAGCACGCCAGGGACTATTA (SEQ ID NO: 80) BmpA-7CATGACACCGTACCTGCTCTCCCTTCTGACTGGATGCCGGATCTGGGCCGATTTTGTTCGCGCCCCGCCCAAGCACGCCAGGGACTATTA (SEQ ID NO: 81) BmpA-8CATGACACCGTACCTGCTCTTTCCGCTGGTTCCACGTGGTCCCGCGTAGGTTCGTGTGCGCGCAAAATCCAAGCACGCCAGGGACTATTA (SEQ ID NO: 82) BmpA-9CATGACACCGTACCTGCTCTGCCCCTGCGTGCCGCAGTCAATCACCATGTTGTTATTACGGACTACCTGGAAGCACGCCAGGGACTATTA (SEQ ID NO: 83) BmpA-10CATGACACCGTACCTGCTCTCCGGTACGATAGGGGTTGAGTTGGACACACTGCCTGGTTAAATTGTGCAGAAGCACGCCAGGGACTATTA (SEQ ID NO: 84)

In certain embodiment, the present invention includes aptamerscomprising a nucleotide sequence that is at least 50% identical to anucleotide sequence selected from the group consisting of: SEQ ID Nos.67-84, or any homologue thereof, or any combinations thereof. In certainembodiments, the aptamers comprise a nucleotide sequence that is atleast 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%,96%, 97%, 98%, 98.1%, 98.2%, 98.3%, 98.4%, 98.5%, 98.6%, 98.7%, 98.8%,98.9%, 99%, 99.1%, 99.2%, 99.3%, 99.4%, 99.5%, 99.6%, 99.7%, 99.8%, or99.9%, identical to a nucleotide sequence selected from the groupconsisting of: SEQ ID Nos. 67-84, or any homologue thereof, or anycombinations thereof.

REFERENCES

-   (1) Hofmann, H. P. et al. RNA 1997, 3, 1289.-   (2) Ellington, A. D. et al. Nature 1990, 346, 818.-   (3) Geiger, A. et al. Nucleic Acids Res. 1996, 24, 1029.-   (4) Strehlitz, B. et al. Bioanal. Rev. 2012, 4, 1.-   (5) Williams, K. P. et al. Proc. Natl. Acad. Sci. 1997, 94, 11285.-   (6) Purschke, W. G. et al. Nucleic Acids Res. 2003, 31, 3027.-   (7) Mie, M. et al. Appl. Biochem. Biotechnol. 2013, 169, 250.-   (8) Ohuchi, S. Biores Open Access 2012, 1, 265.-   (9) Ikanovic, M. et al. J. Fluoresc. 2007, 17, 193.-   (10) Kim, Y. S. et al. Anal. Biochem. 2013, 436, 22.

D. Raman-Active Marker Molecules

Raman-active markers are chemical moieties covalently attached to one ormore aptamers. The markers are typically fluorescent markers that havebeen incorporated into phosphoramidites (monomers used to build DNAsequences on solid support) designed for incorporation into syntheticoligonucleotides. There are also a second class of Raman active markers,which are not as large (and therefore not as likely to affect theinteractions of the aptamer and target) such as alkynes, azides etc.(Yamakoshi (2012) J Am Chem Soc 134: 20681-9) that can also beincorporated into synthetic oligo molecules as phosphoramidites. UsefulRaman active markers include but are not limited to, Cy3, TAMRA,Texas-Red (TR), Cy3.5, Rhodamine 6G, Cy5. Because so many Raman-activemarkers are available, many more than with fluorescent dyes, since minorchemical modification of a Raman marker molecule can lead to a new onewith a different Raman spectrum even though the two molecules exhibitvirtually indistinguishable fluorescence spectra Kneipp et al. Chem.Rev. 99, 2957 (1999); Graham et al., Angew. Chem. Int. Ed. 39, 1061(2000). This allows multiplexing by using Raman-active markers withunique spectral signatures for each aptamer or set of aptamers for agiven target.

Non-limiting examples of Raman-active markers that can be used for Ramanspectroscopy include alkynes, azide, TRIT (tetramethyl rhodamineisothiol), NBD (7-nitrobenz-2-oxa-1,3-diazole), Texas Red (TR) dye,phthalic acid, terephthalic acid, isophthalic acid, cresyl fast violet,cresyl blue violet, brilliant cresyl blue, para-aminobenzoic acid,erythrosine, biotin, digoxigenin,5-carboxy-4′,5′-dichloro-2′,7′-dimethoxy fluorescein,5-carboxy-2′,4′,5′,7′-tetrachlorofluorescein, 5-carboxyfluorescein,5-carboxy rhodamine, 6-carboxyrhodamine, 6-carboxytetramethyl aminophthalocyanines, azomethines, cyanines, xanthines, succinylfluoresceins,aminoacridine, quantum dots, carbon nanotubes and fullerenes. These andother Raman labels may be obtained from commercial sources (e.g.,Molecular Probes, Eugene, Oreg.; Sigma Aldrich Chemical Co., St. Louis,Mo.; Glen Research, Sterling, Va.) and/or synthesized by methods knownin the art.

Polycyclic aromatic compounds may function as Raman-active markers, asis known in the art. The skilled artisan will realize that the Ramanlabels used should generate distinguishable Raman spectra and may bespecifically bound to or associated with different types of aptamers.

Labels or Raman-active markers may be attached directly to the aptamersor may be attached via various linker compounds during solid phase DNAsynthesis. Cross-linking reagents and linker compounds of use in thedisclosed methods are known in the art Raman labels that containreactive groups designed to covalently react with other molecules, suchas the aptamers, are commercially available (e.g., Thermo Scientific,Eugene, Oreg.). Methods for preparing labeled analytes are known (e.g.,U.S. Pat. Nos. 4,962,037; 5,405,747; 6,136,543; 6,210,896).

E. Target Definition

As used herein, the term “target” or “analyte” are used interchangeablyand mean any atom, chemical, molecule, compound, composition oraggregate of interest for detection and/or identification. Non-limitingexamples of analytes include an amino acid, peptide, polypeptide,protein, glycoprotein, lipoprotein, nucleoside, nucleotide,oligonucleotide, nucleic acid, sugar, carbohydrate, oligosaccharide,polysaccharide, fatty acid, lipid, hormone, metabolite, cytokine,chemokine, receptor, neurotransmitter, antigen, allergen, antibody,substrate, metabolite, cofactor, inhibitor, drug, pharmaceutical,nutrient, prion, toxin, poison, explosive, pesticide, chemical warfareagent, biohazardous agent, radioisotope, vitamin, heterocyclic aromaticcompound, carcinogen, mutagen, narcotic, amphetamine, barbiturate,hallucinogen, drugs of abuse, waste product, gunshot residue, and/orcontaminant.

In certain embodiments, the analyte or target is present in species ofthe family Borrelia. In certain embodiments, the analyte is a B.burgdorferi surface protein selected from OspA, OspB, OspC, BmpA, orcombination thereof. OspA, OspB, OspC, and BmpA are outer surfaceproteins found on B. burgdorferi; and they are differentially expressedin humans or within the tick anatomy. OspA and OspB appear to beessentially for survival of B. burgdorferi in the tick, and OspC isessential for infection of the mammalian host by infected ticks.

F. Biological Sample

As used herein, the term “biological sample” may include, but is notlimited to blood, blood products, serum, plasma, other blood fractions,tissue, tissue extracts, urine, cerebrospinal fluid, saliva, feces,skin, hair, cheek tissue, organ tissue, breath, pleural fluid, sweat, orsputum.

As used herein, a “sample collection apparatus” or “sample collectiondevice” may include, but are not limited to, a swab or other matrix (afilter paper, cotton, pad, or foam), dipstick, test strip, cup,cartridge, capillary, or tube. The biological sample collected orcontained therein may be reconstituted, for example in water or asuitable buffer. In certain embodiments, the biological sample does notrequire reconstitution. The sample collection device may be color coded,e.g. using a dye, detectable tag or bar code, to indicate whether thedevice is a positive or negative control. In certain embodiments, thematrix or collection device may be made of bonded polyolefin fiber suchas Bonded Polyolefin Fibre, Glass Fiber, cellulose, cotton,polyethylene, nylon, natural macromolecules, polyvinyl sulfone, silica,glass fiber, glass fiber with binder, cellulose acetate, ornitrocellulose (NC). The “sample collection apparatus” or “samplecollection device” may comprise a housing, wherein said housing maycomprise a material suitably adapted for sample collection, such asplastic, and the like.

The sample collection device may further comprise an enzyme or protease,protein, a compound or preservative for processing the biologicalsample, increasing shelf-life, a chemical stabilizer, diluent, buffer,additive, detergent, lipid, sugar, carbohydrate, or any combinationthereof. For example, the enzyme or protease may solubilize thebiological or test sample. For example, the enzyme may be, but notlimited to, mucin. The protein may be, but not limited to, bovine serumalbumin. The preservative may be, but not limited to, sodium azide.Other additives and stabilizers may include, but not limited to,di-sodium hydrogen orthophosphate anhydrous, potassium dihydrogenorthophosphate, d-Mannitol, or any combination thereof.

G. Raman Detectors or Instruments

Analytes may be detected and/or identified by known methods of SERSRaman spectroscopy or other appropriate Raman spectroscopic techniques,such as tip enhanced raman scattering (TERS) and single molecule Ramanscattering (SMERS). Variations on surface enhanced Raman spectroscopy(SERS), surface enhanced resonance Raman spectroscopy (SERRS),hyper-Raman spectroscopy and coherent anti-Stokes Raman spectroscopy(CARS) have been disclosed. In SERS and SERRS, the sensitivity of theRaman detection is enhanced by a factor of 10⁶ or more for moleculesadsorbed on roughened metal surfaces or nanoparticles, such as silver,gold, platinum, copper or aluminum surfaces. By adding the Raman-activemarker to the aptamers in accordance with this invention, there isfurther and stronger signal enhancement; further the addition of themarker allows identification of the target through the known anddistinct spectral characteristics of the marker that is expected to beunique when compared to the matrix surrounding it, or the unmodifiedaptamer. One useful method of SERS detection is described in U.S. PatentPublication No. 2013/0107254.

A non-limiting example of a Raman detection unit is disclosed in U.S.Pat. No. 6,002,471. An excitation beam is generated by either afrequency doubled Nd:YAG laser at 532 nm wavelength or a frequencydoubled Ti:sapphire laser at 365 nm wavelength. Pulsed laser beams orcontinuous laser beams may be used. The excitation beam passes throughconfocal optics and a microscope objective, and is focused onto theRaman active complex containing one or more analytes. The Raman emissionsignal is collected by the microscope objective and the confocal opticsand is coupled to a monochromator for spectral dissociation. Theconfocal optics includes a combination of dichroic filters, barrierfilters, confocal pinholes, lenses, and mirrors for reducing thebackground signal. Standard full field optics can be used as well asconfocal optics. The Raman emission signal is detected by a Ramandetector, comprising an avalanche photodiode interfaced with a computerfor counting and digitization of the signal.

Another example of a Raman detection unit is disclosed in U.S. Pat. No.5,306,403, including a Spex Model 1403 double-grating spectrophotometerwith a gallium-arsenide photomultiplier tube (RCA Model C31034 or BurleIndustries Model C3103402) operated in the single-photon counting mode.The excitation source comprises a 514.5 nm line argon-ion laser fromSpectra Physics, Model 166, and a 647.1 nm line of a krypton-ion laser(Innova 70, Coherent).

In certain embodiments, the typical incident wavelengths are about 488,about 514.5, about 532 nm, about 632.8, about 785 nm and about 1064 nm.

Alternative excitation sources include a nitrogen laser (Laser ScienceInc.) at 337 nm and a helium-cadmium laser (Liconox) at 325 nm (U.S.Pat. No. 6,174,677), a light emitting diode, an Nd:YLF laser, and/orvarious ions lasers and/or dye lasers. The excitation beam may bespectrally purified with a bandpass filter (Corion) and may be focusedon the Raman active complex using a 6× objective lens (Newport, ModelL6X). The objective lens may be used to both excite the analytes and tocollect the Raman signal, by using a holographic beam splitter (KaiserOptical Systems, Inc., Model HB 647-26N18) to produce a right-anglegeometry for the excitation beam and the emitted Raman signal. Aholographic notch filter (Kaiser Optical Systems, Inc.) may be used toreduce Rayleigh scattered radiation. Alternative Raman detectors includean ISA HR-320 spectrograph equipped with a red-enhanced intensifiedcharge-coupled device (RE-ICCD) detection system (PrincetonInstruments). Other types of detectors may be used, such asFourier-transform spectrographs (based on Michaelson interferometers),charged injection devices, photodiode arrays, InGaAs detectors,electron-multiplied CCD, intensified CCD and/or phototransistor arrays.

Any suitable form or configuration of Raman spectroscopy or relatedtechniques known in the art may be used for detection of the complexesof the invention, including but not limited to normal Raman scattering,resonance Raman scattering, surface enhanced Raman scattering, surfaceenhanced resonance Raman scattering, coherent anti-Stokes Ramanspectroscopy (CARS), stimulated Raman scattering, inverse Ramanspectroscopy, stimulated gain Raman spectroscopy, hyper-Ramanscattering, molecular optical laser examiner (MOLE) or Raman microprobeor Raman microscopy or confocal Raman microspectrometry,three-dimensional or scanning Raman, Raman saturation spectroscopy, timeresolved resonance Raman, Raman decoupling spectroscopy or UV-Ramanmicroscopy.

In certain embodiments, the present invention discloses methods fortesting whether a point of care testing apparatus, such as a portableRaman detector, is properly functioning. The method comprises the stepsof: (a) contacting a sample collection device with water, a bufferedsolution, or biological sample; (b) inserting the sample collectiondevice into a point of care testing apparatus; and (c) detecting thepresence of the positive or negative control, wherein the presence ofthe positive or negative control indicates that the point of caretesting apparatus is properly functioning. In certain embodiments, astandard test cartridge for calibration may be used to test if the pointof care testing apparatus is properly functioning.

H. Other Technologies

Current methods to detect the presence of B. burgdorferi in ticks useimmunological methods to detect bacterial proteins present in tickhomogenates. Typically, these tests are time consuming, requiringsignificant technical skill and training to conduct. Often, ticks aresent into testing laboratories, and it can take weeks for results to bereturned; many individuals will have already shown Lyme disease symptomsbefore they receive the results for the tick testing.

The current accepted method for detecting Lyme disease in humans isbased on a CDC-approved, two-tiered serological protocol using wholecell homogenates as targets for the detection of human anti-B.burgdorferi antibodies. The first tier tests typically use an ELISA orrelated assay to detect anti-Lyme IgM or IgG antibodies, and positive orequivocal results will trigger the use of the second test, an IgM or IgGimmunoblot using B. burgdorferi extracts to detect bands deemed specificfor B. burgdorferi. A number of problems exist with these serologicalassays: 1) poor sensitivity; 2) very low levels anti-B. burgdorferiantibodies in the first 4-6 weeks of infection; 3) poor anti-B.burgdorferi responses in some individuals; 4) subjective interpretationof immunoblots; and 5) the inability to diagnose B. burgdorferiinfections in antibody treated or re-infected individuals. Because thistest detects antibody production and not the pathogen directly, the testcannot detect infections within 3 weeks of infection. Additionally, thetest has a specificity >80%, but a sensitivity of only 50-60%. Currentestimates in the US suggest that approximately 300,000 human Lymedisease tests are ordered, though many clinicians do not order the testdue to the low sensitivity. The low rates at which Lyme disease isreported is in part due to the poor quality of the diagnostic toolcurrently available to clinicians.

In addition to serologically-based assays, a few other new assays havestarted to appear in the literature and in the market place. Ametabolomics approach to Lyme diagnostics was recently described(Mollins, C. R. et al. Clin Infect Dis. 2015 Mar. 11.). This method isin early stage development and requires instrument-intensive,technically advanced GC-MS analysis, making point-of-care (POC)detection unlikely. Ceres Nanosciences has developed ananoparticle-based assay that concentrates Lyme antigens from urine anddetects their presence using standard immunoassay technology (Douglas,T. A. et al. Biomaterials. 2011 February; 32(4):1157-66;www.ceresnano.com). Although this assay directly detects Lyme antigensinstead of anti-B. burgdorferi antibodies, the assay uses immunoassaytechnology to detect Lyme antigens and requiring >4 hrs to conduct, alsomaking POC detection difficult.

A variety of other Lyme disease diagnostic assays have been described inthe literature. Some include direct DNA amplification-based assays,culture-based assays, and other direct Lyme antigen detection methods,such as a transistor-based assay that measures Lyme antigen binding toantibody modified carbon nanotubes (Lerner, M. B. et al. BiosensBioelectron. 2013 Jul. 15; 45:163-7). Many of these assays are in earlystages of development or have yielded results inferior to the currentCDC-approved two-tiered immunoassay.

The present invention has several advantages of the methods describedabove, including: 1) DNA aptamers provide a high affinity, flexible, andeasily generated and modified alternative to anti-Lyme antigenantibodies used in other detection systems; 2) the nanoparticle-basedantigen binding allows for easy and rapid binding and separation of theantigens from bulk blood, serum or plasma; 3) the 10⁶ to 10¹⁴-foldsignal amplification of SERS allows for ultra-sensitive Lyme antigendetection; 4) the proprietary placement Raman labels along the DNAaptamers dramatically improves specificity and sensitivity of theSERS-based detection; and 5) the entire detection method, from antigenbinding to SERS detection takes <30 minutes, making it highlyappropriate for POC diagnostic applications.

As can be appreciated from the disclosure above, the present inventionhas a wide variety of applications. The invention is further illustratedby the following examples, which are only illustrative and are notintended to limit the definition and scope of the invention in any way.

EXEMPLIFICATIONS I. Reagents and Methods for Detecting InfectiousDiseases (Examples 1-4) Example 1 Aptamer Selection for Oxytocin (OT)and oxytocin-gly-lys-arg (OT-GKR)

Aptamers against OT and OT-GKR were selected by adapting the selectionprocess described by Hoon et al.¹ In this method, biotin-OT andbiotin-OT-GKR are bound to magnetic, biotinylated microspheres, andthese microspheres act as an easily collected substrate to separate DNAoligonucleotides bound to the peptide hormones from unbound oligos. Theoligonucleotide pool used as the aptamer selection library consisted of40 random nucleotides flanked by short stretches of constant DNAsequences which can act as primer sites necessary for PCR amplificationof the aptamer library following aptamer selection. The sequence of theaptamer library was:

(SEQ ID NO: 1) 5′-ACACTCTTTCCCTACACGACGCTCTTCCGATCT-[N]₄₀-AGATCGGAAGAGCACACGTCTGAACTCCAGTCAC-3′Flanking regions for this oligo were chosen to be complementary to theadapter sequences for next generation sequencing (NGS) of DNA on theIllumina MiSeq sequencing platform. Briefly, the aptamer selectionprocess is as follows:

-   -   0.1 ml of Dynabeads m270 are used for each round of selection        against a target, and 0.1 ml Dynabeads are used for each        negative selection    -   Dynabeads are washed 3 times with PBS    -   Dynabeads for positive selection are incubated (with constant        rotation) with 100 pmol biotin-OT or biotin-OT-GKR in PBS for 30        min at room temperature    -   Peptide bound Dynabeads are washed 3 times with PBS    -   All Dynabeads samples (OT, OT-OKR, and 2 samples for negative        control) are washed 2 times with PBS+0.1% BSA    -   Aptamer Selection Library DNA (in the first round of selection        15 nmol of DNA is used for each selection; in subsequent        selection rounds 90% of the aptamer library collected from the        previous round is used) is incubated at 95° C. for 5 min, 0° C.        for 5 min, and room temperature for 5 min    -   DNA is added to the negative control Dynabeads and incubated at        room temperature for 30 min with constant rotation    -   The supernatant from the negative selection Dynabeads is        transferred to either the OT or OT-GKR bound Dynabeads—samples        are incubated at room temperature for 30 min with constant        rotation    -   Supernatant is removed from the Dynabeads and the Dynabeads are        washed 3 times with PBS+0.1% BSA and 1 time with PBS    -   Dynabeads with bound aptamer library DNA is stored at −20° C.        until ready for PCR amplification and analysis    -   Following PCR analysis, approximately 20% of each sample is sent        for NGS DNA sequencing on an Illumina MiSeq instrument and the        remaining fraction was used to generate single stranded DNA        (ssDNA) to be used in the next round of aptamer selection    -   ssDNA is generated using asymmetric PCR followed by Lambda        Exonuclease digestion of the reverse complement strand of DNA

High Throughput DNA Sequencing

Following aptamer selection, oligonucleotides bound to the Dynabeads arePCR-amplified using primers that add the Illumina TruSeq UniversalAdapter sequence to the 5′-end of the oligo and a TruSeq Indexed primercontaining a 6-base barcode to the 3′-end of the oligo. Table 1.3 showsfour samples that are generated for NGS analysis. Underlined sequencesrepresent the 6 base barcodes that are required to distinguish pooledsamples on the DNA sequencing procedure.

TABLE 1.3 Reverse PCR Primer Oligonucleotides ShowingTruSeq Index Barcodes Index Sample No. Primer Sequence Oxytocin 6CAAGCAGAAGACGGCATACGAGATATTGGCGTGA CTGGAGTTCAGACGTGTGCTCTTCCGATCT (SEQ ID NO: 2) Oxytocin- 7 CAAGCAGAAGACGGCATACGAGATGATCTGGTGA GKRCTGGAGTTCAGACGTGTGCTCTTCCGATCT (SEQ ID NO: 3) Negative 12CAAGCAGAAGACGGCATACGAGATTACAAGGTGA ControlCTGGAGTTCAGACGTGTGCTCTTCCGATCT (SEQ ID NO: 4) Aptamer 17CAAGCAGAAGACGGCATACGAGATCTCTACGTGA LibraryCTGGAGTTCAGACGTGTGCTCTTCCGATCT (SEQ ID NO: 5)Following PCR amplification, the samples are sequenced. Approximatelyequimolar amounts of the four samples are pooled and run on a singlelane of the MiSeq sequencer, which is capable of sequencing 20,000,000DNA strands in an overnight analysis. The instrument is set to readsequences up to 50 bp in length, allowing the random sequence to bedetermined in its entirety with every sequencing reaction. Sequencesassociated with each sample are identified by the 6 base barcode addedto the aptamer by PCR prior to the sequencing reaction. Bioinformatics

To analyze the sequence data generated by the Illumina MiSeqhigh-throughput DNA sequencer, analytical tools for aptamer analysiswere developed by adapting the methods described in Latulippe et al.⁴

First, the data from the Illumina MiSeq DNA sequencer are sorted by DNAbarcode and mathematically filtered to remove artifactual sequences,such as sequences that contained long stretches of homopolymers. Thenall sequences from a single sample are compared and all exact matcheswithin a sample are counted and sorted by the number of times thesequence occurs. The most common 5000 sequences from each sample areexported into Microsoft Excel. The number of occurrences of a specificsequence is normalized for each sample against the total number ofsequences that are generated for each sample (i.e. OT, OT-GKR, ornegative control). The most commonly occurring samples from the OTselection are compared with sequences from the OT-GKR selection and theNegative control selection, and sequences that are unique to the OTselection are highlighted. These sequences are labeled as potential OTaptamers. The same analysis is used to analyze the OT-GKR selection togenerate aptamers that are potential OT-GKR aptamers.

Table 1.4 shows the results from these analyses. Of the top 30sequences, only one oxytocin aptamer sequence is both unique to the OTlibrary and absent from the top 5000 negative control sequences (oligonumber followed by R). Two additional sequences were unique to OT andwere only rarely (fewer than 5 times out of −5×10⁶ sequences) found inthe negative control sequences (oligo number followed by Y). For the top30 OT-GKR sequences, two unique sequences were found and one sequencewas found that was unique to OT and was only rarely found in thenegative control sequences.

Based on the results shown in Table 1.4, the following oligonucleotidesare synthesized and tested for OT and OT-GKR binding:

Oxytocin (SEQ ID NO: 6) 5′-ATGCAAATTAGCATAAGCAGCTTGCAGACCCATAATGTC-3′(SEQ ID NO: 7) 5′-ATAGTGTTATTAATATCAAGTTGGGGGAGCACATTGTAG-3′(SEQ ID NO: 8) 5′-CTTGTTTACGAATTAAATCGAAGTGGACTGCTGGCGGAA-3′Oxytocin-GKR (SEQ ID NO: 9)5′-TAAACGTGACGATGAGGGACATAAAAAGTAAAAATGTCT-3′ (SEQ ID NO: 6)5′-ATGCAAATTAGCATAAGCAGCTTGCAGACCCATAATGTC-3′ (SEQ ID NO: 11)5′-AGTTGCCATACAAAACAGGGTCGCCAGCAATATCGGTAT-3′

TABLE 1.4 Results of Sequence Comparisons of Aptamers Obtained from Illumina MiSeq DNASequencing Oxytocin Oxytocin-GKR No of No of Oligos Oligo SequencesOligos Oligo Sequences 18 (SEQ ID NO: 12) 18 (SEQ ID NO: 39)TAGCCACATAGAAACCAACAGCCATATAACTGGTAGCTTTTTCCTGCTCCTGTTGAGTTTATTGCTGCCGTCATTGCT 17 (SEQ ID NO: 13) 17(SEQ ID NO: 40) CATAATGTCAATAGATGTGGTAGAAGTCGTCATTTGGCGGGTCAGTAGCAATCCAAACTTTGTTACTCGTCAGAAAAT 17 (SEQ ID NO: 14) 17(SEQ ID NO: 41) TAATAACCTGATTCAGCGAAACCAATCCGCGGCATTTAGAAATAGTTGTTATAGATATTCAAATAACCCTGAAACAAA 16 (SEQ ID NO: 15) 16(SEQ ID NO: 42) CACAGTCCTTGACGGTATAATAACCACCATCATGGCGACGGGAGGGTAGTCGGAACCGAAGAAGACTCAAAGCGAACC 16 (SEQ ID NO: 16) 15(SEQ ID NO: 43) GCGGCGGCAAGTTGCCATACAAAACAGGGTCGCCAGCAAGTCATTTGGCGAGAAAGCTCAGTCTCAGGAGGAAGCGGA 16 (SEQ ID NO: 17) 15(SEQ ID NO: 44) TATTTAACTGGCGGCGATTGCGTACCCGACGACCAAAATCGGCGTACGGGGAAGGACGTCAATAGTCACACAGTCCTT 15 (SEQ ID NO: 18) 14(SEQ ID NO: 45) AAGAGCAGAAGCAATACCGCCAGCAATAGCACCAAACATATAATCTCTTTAATAACCTGATTCAGCGAAACCAATCCG 15 (SEQ ID NO: 19)

(SEQ ID NO: 9) CAGCGAAACCAATCCGCGGCATTTAGTAGCGGTAAAGTTTAAACGTGACGATGAGGGACATAAAAAGTAAAAATGTCT 14 (SEQ ID NO: 20) 14(SEQ ID NO: 46) TATGGCTAAAGCTGGTAAAGGACTTCTTGAAGGTACGTTATTAGCTGTACCATACTCAGGCACACAAAAATACTGATA

(SEQ ID NO: 7) 14 (SEQ ID NO: 47)ATAGTGTTATTAATATCAAGTTGGGGGAGCACATTGTAGTTGTTATAGATATTCAAATAACCCTGAAACAAATGCTTA 14 (SEQ ID NO: 21) 13(SEQ ID NO: 48) ATGGAAATGAAGACGGCCATTAGCTGTACCATACTCAGGTGTAGCGAACTGCGATGGGCATACTGTAACCATAAGGCC 13 (SEQ ID NO: 22) 13(SEQ ID NO: 49) CTCTTTAGTCGCAGTAGGCGGAAAACGAACAAGCGCAAGAGCTTACTAAAATGCAACTGGACAATCAGAAAGAGATTG 13 (SEQ ID NO: 23) 13(SEQ ID NO: 50) ACGAAAGACCAGGTATATGCACAAAATGAGATGCTTGCTTCAATAGCAGGTTTAAGAGCCTCGATACGCTCAAAGTCA 13 (SEQ ID NO: 24) 13(SEQ ID NO: 51) CATATAACTGGTAGCTTTAAGCGGCTCACCTTTAGCATCCCGCTTCGGCGTTATAACCTCACACTCAATCTTTTATCA 13 (SEQ ID NO: 25) 13(SEQ ID NO: 52) TGAAACCAACATAAACATTATTGCCCGGCGTACGGGGAATATCAGGGTTAATCGTGCCAAGAAAAGCGGCATGGTCAA 12 (SEQ ID NO: 26)

(SEQ ID NO: 11) TTTAGCCATAGCACCAGAAACAAAACTAGGGACGGCCTCAGTTGCCATACAAAACAGGGTCGCCAGCAATATCGGTAT 12 (SEQ ID NO: 27) 12(SEQ ID NO: 53) TTTAGTCGCAGTAGGCGGAAAACGAACAAGCGCAAGAGTCACCAAACATAAATCACCTCACTTAAGTGGCTGGAGACA 12 (SEQ ID NO: 28) 12(SEQ ID NO: 54) AAGCACCTTTAGCGTTAAGGTACTGAATCTCTTTAGTCGTCCATATCTGACTTTTTGTTAACGTATTTAGCCACATAG 12 (SEQ ID NO: 29) 12(SEQ ID NO: 55) ATTCTTTAGCTCCTAGACCTTTAGCAGCAAGGTCCATATAATAATGTTTATGTTGGTTTCATGGTTTGGTCTAACTTT 12 (SEQ ID NO: 30) 12(SEQ ID NO: 56) ATTGGTATCAGGGTTAATCGTGCCAAGAAAAGCGGCATGACGTTAACAAAAAGTCAGATATGGACCTTGCTGCTAAAG 12 (SEQ ID NO: 31) 12(SEQ ID NO: 57) CAGATATTGAAGCAGAACGCAAAAAGAGAGATGAGATTGACGTTGGCTGACGACCGATTAGAGGCGTTTTATGATAAT 11 (SEQ ID NO: 32) 12(SEQ ID NO: 58) AAAAACGATAAACCAACCATCAGCATGAGCCTGTCGCATCATGGTGGCGAATAAGTACGCGTTCTTGCAAATCACCAG

(SEQ ID NO: 6) 12 (SEQ ID NO: 59)ATGCAAATTAGCATAAGCAGCTTGCAGACCCATAATGTCCGGGCAATAATGTTTATGTTGGTTTCATGGTTTGGTCTA 11 (SEQ ID NO: 33) 12(SEQ ID NO: 60) CAGTAGGCGGAAAACGAACAAGCGCAAGAGTAAACATAGGCGATGGGCATACTGTAACCATAAGGCCACGTATTTTGC 11 (SEQ ID NO: 34) 11(SEQ ID NO: 61) CCTCACTTAAGTGGCTGGAGACAAATAATCTCTTTAATAAACAAAAAGTCAGATATGGACCTTGCTGCTAAAGGTCTA

(SEQ ID NO: 8) 11 (SEQ ID NO: 62)CTTGTTTACGAATTAAATCGAAGTGGACTGCTGGCGGAAACGCGGCACAGAATGTTTATAGGTCTGTTGAACACGACC 11 (SEQ ID NO: 35) 11(SEQ ID NO: 63) GAAGTGCCAGCCTGCAACGTACCTTCAAGAAGTCCTTTAATGGTGGCGAATAAGTACGCGTTCTTGCAAATCACCAGA 11 (SEQ ID NO: 36) 11(SEQ ID NO: 64) GCATCATCTTGATTAAGCTCATTAGGGTTAGCCTCGGTACCGCCAGTTAAATAGCTTGCAAAATACGTGGCCTTATGG 11 (SEQ ID NO: 37) 11(SEQ ID NO: 66) GGATTTGAGAATCAAAAAGAGCTTACTAAAATGCAACTGTAACAGATACAAACTCATCACGAACGTCAGAAGCAGCCT 11 (SEQ ID NO: 38)

(SEQ ID NO: 10) TGACCAGCAAGGAAGCCAAGATGGGAAAGGTCATGCGGCTTTTAAAGCGCCGTGGATGCCTGACCGTACCGAGGCTAA Notes = :

Example 2 Synthesis of, and Oxytocin Detection with, Aptamer-ModifiedSilver Nanoparticles (Ag-NPs)

Synthesis of the Ag NPs were performed using the literature proceduredescribed by Leona,⁵ in which the NPs were synthesize through reductionof silver sulfate in the presence of glucose and citrate. Briefly, thesynthetic method involved the precipitation of silver sulfate from anaqueous solution of silver nitrate through the addition of sulfuricacid. A 5×10⁻⁴ M aqueous solution of the freshly precipitated silversulfate salt was prepared, and 12.5 mL of this solution was mixed with1% solutions of citrate (1 mL) and glucose (500 μL). The solution washeated using a microwave digestion system (Anton Parr Multiwave 3000),using three heating programs in which the temperature of the solutionwas ramped to 120° C. over 30 seconds, and held at this temperature for30, 60 or 90 seconds. The NPs were then purified by centrifugation for15 minutes, after which the supernatant was removed and replaced withdistilled deionized water to reduce the citrate concentration.

Each of the temperature programs resulted in Ag NPs that demonstratedspectroscopic characteristics that compared favorably with thosereported in the literature (FIG. 5, Panel A and Panel B).⁵′⁶ Theabsorption maximum was found at 401 nm, with a full maximum at half withvalue of 60 nm, which compares favorably with the 56 nm value from theliterature.⁵ Since the plasmon resonance is intimately related to theparticle size, the NPs are determined to be 20 to 25 nm based on thesedata by comparison with literature.⁷

The absorption spectra of the NPs were collected three weeks after thesynthesis was initially performed to demonstrate the stability of theNPs. Absorption spectra of NPs stored in the reaction mixture beforecentrifugation, with high citrate and glucose concentrations, and thosethat had been centrifuged on the day the NPs had been synthesized, tolower citrate concentrations, were compared (FIG. 5, Panel A). Thesimilarity of the absorption spectra with each other, and with thespectrum collected the day of the synthesis, indicates themicrowave-based synthetic method produce stable Ag NPs capable ofserving as the basis of a robust method for oxytocin detection.

The ability of these Ag NPs to produce Raman enhancement was thendemonstrated using a model SERS active compound, mercaptophenol (MCP).MCP was used in these studies for two important reasons: it produces asignature Raman spectrum that allows the efficacy of the NPs as SERSsubstrates to be determined, and it interacts with the Ag NPs through athiol linkage analogous to the one used to modify the surface of Ag NPswith thiolated aptamers. The spectrum of a 200 μM solution of MCPcollected in the presence of the Ag NPs and a 100 μM solution ofpotassium nitrate was compared to the spectrum of the same solution inthe absence of the NPs, demonstrating a five order of magnitudeenhancement provided by the presence of the NPs (FIG. 2, Panel B).

Building on the successful synthesis of the Ag NPs were performed usingthe literature procedure described by Leona,⁵ and demonstration of theability of the synthesized Ag NPs to produce a five order of magnitudeincrease in spectral intensity, this facet of the research focused onthe modification of Ag and Au NPs with thiolated DNA. Thiolated DNA istypically synthesized using a disulfide moiety, requiring the reductionof the disufide to the thiol or dithiol moiety in anticipation ofmodifying NP surfaces with the oligonucleotide. In order to perform thereduction, three methods were applied: the first involved the reductionof the disulfide with: dithiothreitol (DTT) under basic conditions,⁸tris(2-carboxyethyl)phosphine (TCEP) under acidic conditions,⁹ or simplyexposing the disulfide terminated DNA to the NPs to facilitate reductionof the disulfide.¹⁰

Investigation of the efficacy of the surface modification were performedusing DNA modified with oligonucleotides that are terminated with eithermono- or dithiol-moieities. The efficacy of this approach was monitoredusing absorption spectroscopy. Surface modification of the NPs generallyinvolves incubation of the NPs in the presence of thiol-modified DNA for16-24 hrs., followed by slow addition of salt to facilitate an increaseof DNA concentration at the NP surface. The slow addition of salt,typically in 50 mM increments over the span of 2-5 days to a finalconcentration of 300 mM, is intended to increase the concentration ofDNA at the NP surface by balancing the charge of the phosphate moietiesin the DNA backbone, reducing repulsion. Salt conditions may be adjustedto produce DNA-modified NP SERS substrates.

Comparison of the monothiol- and dithiol-terminated oligonucleotides,involved investigation of two parameters in the surface modificationwith the dithiol-terminated oligonucleotides: the reducing agent used toactivate the disulfide modifier, and the rate and concentration of thesalt to increase the concentration of DNA at the NP surface. Theefficacy of the surface modification was monitored by measuring theretention of the surface plasmon absorption. The initial step, in whichthe disulfide modified DNA was reduced to produce the dithiol terminuscapable of ligating the Ag NP surface, was performed in the presence ofdithiothretol (DTT),⁹ (tris(2-carboxyethyl)phosphine) (TCEP),⁸ and inthe absence of reducing agent by direct exposure of the disulfide to theNP suspension.¹⁰

As illustrated in FIG. 2, Panel C, the plasmon is stable after exposureto both dithiol-terminated DNA and the addition of 25 mM NaCl.Increasing the concentration to 50 mM salt results in the first shift ofthe plasmon absorption to lower energy. Further addition of salt,bringing the salt concentration to a total of 100 mM, results in rapidloss of the plasmon indicative of NP aggregation.

Each of the approaches were performed in parallel and monitored usingabsorption spectroscopy (FIG. 2, Panel D). While the intensity of thesurface plasmon indicated that each of the reduction methods resulted insuccessful addition of the dithiol-modified DNA, there were significantdifferences noted when sodium chloride was added to increase theconcentration of DNA at the NP surface. Addition of sodium chloride in25 mM aliquots to Ag NPs modified in the absence of reducing agent, orin the presence of TCEP did not significantly decrease the intensity ofthe plasmon absorption, however NPs that had been exposed to thedithiol-modified DNA reduced with DTT resulted in a significant decreasein the plasmon absorption. A comparison of the changes in plasmonabsorption intensity upon addition of sodium chloride to NPs modified inthe presence of various disulfide reducing conditions (FIG. 2, Panel E),confirms that NPs prepared either with TCEP or in the absence ofreducing agents results in the best retention of the Ag NP plasmon.

The surface modification of the NPs was confirmed in two ways: first, acontrol experiment in which NPs that had not been exposed todithiol-terminated DNA was exposed to the same sodium chloride additionconditions as the DNA modified NPs. This resulted in significant loss ofplasmon intensity upon addition of 25 mM sodium chloride (FIG. 2, PanelE). The second demonstration of the surface modification exploited thepresence of the fluorescent FAM marker present at the 5′-terminus.Following the protocol established by Mirkin and co-workers, theFAM-modified DNA was removed from the surfaces of the Ag NPs by exposingthe DNA-modified NPs to a 0.1 M solution of DTT.¹¹ Followingcentrifugation of the sample to separate the Ag from the supernatant,the flourescence of the solution was measured upon irradiation with 488nm light (FIG. 2, Panel F). The resulting emission spectrum demonstratesthe successful modification of the NP surface with the dithiol-modifiedDNA. The relative intensities of the emission spectra suggest thesurface modification is most effective in the absence of reducing agent,confirming what was observed in the absorption study.

The methods developed herein facilitate the reliable modification ofRaman active Ag NPs with mono-thiol or dithiol-terminated aptameroligonucleotides. Dithiol-terminated oligos produce NPs that are morestable than the mono-thiol terminated oligos, and may be employed in allsubsequent optimization of surface modification procedures. Thedithiol-modified DNA aptamer proxy was also modified with a fluorescentmarker, FAM, at the 5′-terminus. The disulfide modifier has been shownto be more effective at anchoring DNA at the NP surface,¹⁰ andFAM-terminated DNA has been used to demonstrate the presence of DNA atNP surfaces through fluorescence experiments.¹¹ Based on these results,Ag NPs prepared in the absence of reducing agent and with 50 mM NaCladded to increase the concentration of DNA at the NP surface were usedin all subsequent Raman studies.

Example 3 Sample Preparation for User Ease and Increased Sensitivity

Bringing NPs into close proximity to form “hot spots” through theaddition of common inorganic salts is well suited to this application.The addition of salt has been suggested to result in the aggregation ofNPs in solution, with the resulting proximity of the NPs resulting inthe “sharing” of NP plasmons, increasing enhancement of the vibrationalspectrum beyond that observed in the presence of individual, SERS-activeNPs.

To illustrate the effect of salt on the efficacy of the NPs to producethe Raman scattering enhancement, the effect of adding 100 μM solutionsof two common salts, potassium nitrate and sodium chloride, wasinvestigated. As shown in FIG. 3, Panel A, the addition of both saltsresulted in an increase of an order of magnitude of the spectralintensity, with the potassium nitrate producing spectral intensity 60%higher than that produced upon introduction of sodium chloride. Theseresults formed the basis of the methods established in this effort.

Based on the success of the DNA modification method development, Ramaninterrogations of the resulting materials were interrogated using 632.8nm and 785 nm incident wavelengths, revealing the presence of distinct,reproducible spectral features suggestive of a discrete molecule such asthe fluorescent FAM incorporated into our sequence at the 5′ terminus.The ability to observe features indicative of a dye marker in thepresence of oligonucleotides has been demonstrated by Mirkin andco-workers for multiple dyes, and has used this for multiplexing basedon the intense, distinctive features associated with the dyes.¹²

The first study was performed to determine which salt additiveintroduced just prior to spectral analysis provided the largest spectralfeatures. As illustrated in FIG. 3, Panel B, the addition of KNO₃produces a higher intensity Raman feature, likely due to “hot spot”formation, leading to a synergistic increase in spectral intensity. Inkeeping with this observation, spectral features observed with the AgNPs modified with 5′-FAM terminated DNA were most intense in thepresence of KNO₃.

The strong spectral features demonstrated by FAM provide the opportunityfor development of a method for detection of oxytocin. The sharp,intense spectral features of 5′-FAM rivals the intensity ofmercaptophenol (MCP), and provides spectral features which can be usedas a proxy for the analytical target. The interaction of theFAM-modified aptamer with the aptamer target induces changes in theintensity of the FAM-derived spectral features.

To produce a Ag NP surface monolayer in which both the FAM- andMCP-derived spectral features were observed, the FAM DNA-modified Ag NPswere exposed to varying concentrations of MCP. Mixed monolayers at Ag NPsurfaces composed of FAM-modified and MCP concentrations ranging from 4mM to 4 μM were investigated, with the presence of both MCP and FAMspectral features observed most effectively with a concentration of 4 μMMCP (FIG. 3, Panel C). The mixed MCP/FAM DNA monolayer at the NP surfacerevealed demonstrated distinct, easily differentiable spectral featuresfrom MCP and FAM (FIG. 3, Panel D). Features at 714 cm⁻¹ and 1050 cm⁻¹are derived from the FAM dye found at the 5′-terminus of thedithiol-modified DNA, whereas the features at 613 cm⁻¹, 1010 cm⁻¹, 1080cm⁻¹ and 1175 cm⁻¹ are contributions from the MCP monolayer. Thepresence of these two distinct groups of features suggests these can beused for ratiometric quantitation, where FAM modified DNA-derivedfeatures will change upon introduction of the analyte, while the MCPfeatures will remain constant.

Further investigation of the system with using 785 nm incident lightrevealed a similar set of spectral features. The ability to translatethese spectroscopic features to the lower energy incident light isimportant as the instrumentation using this incident wavelength iscommerically available, relatively inexpensive and readily accessiblefor use by those that have little training in spectroscopy. The spectrumcollected of Ag NPs with surface monolayers composed of 4 mM MCP and theFAM-modified DNA again demonstrated spectral features that could readilybe assigned to the two components of the mixed monolayer. TheFAM-modified DNA contributed features at 305 cm⁻¹, 714 cm⁻¹ and 1050cm⁻¹, whereas the MCP contributed features at 393 cm⁻¹, 636 cm⁻¹, 1010cm⁻¹, 1080 cm⁻¹ and 1175 cm⁻¹. These spectra reveal features that aresimilar to those observed under 633 nm irradiation, as well asadditional features specific to the 785 nm spectra (FIG. 3, Panel E).

A second aspect of the spectral optimization process, the effect ofmixed monolayers composed of Raman-labeled DNA coupled with either MCHor MCP, signal intensity of the monolayer, as well as the effect offurther modification of the surface monolayer was investigated. The workof Lin and co-workers, in which the addition of mercaptohexanol wasdemonstrated to improve the Raman intensity of dye-modified vasopresinusing Raman spectroscopy,¹³ served as the basis for investigation of avariety of surface modifications and new substrates, revealing furtherimprovements in the spectroscopic methods for this application.

The first approach investigated as a method to increase Raman signalintensity involved adding 1-mercaptohexan-6-ol (MCH) to FAM-DNA-modifiedAg NPs. The addition of this molecule to the surface monolayer hasresulted in increased signal intensity in previous Raman-based detectionschemes which rely on apatamers for target recognition.¹³

Three concentrations of MCH: 400 μM, 40 μM and 4 μM, were added to theDNA-modified Ag NPs, revealing a significant increase in the intensityof two FAM-derived spectral features, at 714 cm⁻¹ and 1050 cm⁻¹, at allthree concentrations. The largest increase was observed at the lowestconcentration (FIG. 3, Panel F) and is consistent with previousobservations of thiolated molecules displacing DNA at the NP surface.This phenomenon was exploited to displace FAM-modified DNA from the AgNP surface to demonstrate successful surface modification of the NP(FIG. 2, Panel F).

An investigation parallel to the one studying the effect of MCH on theRaman intensity of Ag NPs with mixed surface monolayers composed ofFAM-DNA, a proxy for the dye-modified aptamer, was performed using MCP,the internal standard against which oxytocin can be quantified, wasexecuted. To identify the optimal surface layer composition, thebaseline corrected intensities of the two largest spectral featuresassociated with the MCP and FAM, 1050 cm⁻¹ and 1080 cm⁻¹ respectively,were compared with varying concentrations of MCH and MCP added to theFAM-DNA modified Ag NPs.

Three concentrations of MCP ranging from 4 to 400 μM, and twoconcentrations of MCH, 4 and 40 μM were tested. This comparison revealedthe largest enhancements of both the MCP and FAM DNA spectral featureswere observed with addition of intermediate concentration of MCP, 40 μM(FIG. 3, Panel F). While it is intuitive that the addition of the lowestconcentration of MCP (4 μM) results in a less intense 1080 cm⁻¹ feature,since there is less MCP available for surface modification, there isalso a decrease in the spectral intensity with an increase in theconcentration of MCP to 400 μM, suggesting competition between thiolatedmolecules at the NP surface. The effect of the competition is alsorefleted in FAM-derived Raman intensities: addition of 400 μM MCPresults in lower intensity, while addition of 4 μM MCP results inminimal changes in the intensity of the 1050 cm⁻¹ feature.

In summary, these investigations provide an understanding of theparameters necessary to optimize SERS-based quantitative determinationof oxytocin levels with aptamer-modified Ag NPs were undertaken. Thefirst involved investigating the effect of adding thiolated molecules tothe surface of FAM-DNA-modified Ag NPs. Two molecular components weretested: MCP, an internal standard molecule and MCH, a molecule capableof filling unoccupied spaces on the NP surface and facilitatingorganization of the molecules at the surface. This study revealed a needfor a balance between the concentrations of the two molecular componentsto provide optimal signal intensity for the FAM and MCP features.

Example 4 Efficacy of Aptamer-Modified NP-Based Oxytocin Detection

Using the conditions established in Example 3, a process foridentification and quantitation from a buffer solution containing thetargets, as well as bovine serum albumin are investigated.Concentrations of vasopressin, as a proxy for oxytocin, is investigatedover a concentration range spanning nM to μM solutions, to both preparea standard curve, and to determine the limit of detection of theproposed approach. The method is developed using the 9-amino acidoxytocin peptide, and extended for use with the 12-amino acidpro-oxytocin.

In preparation for testing the oxytocin-targeting aptamer, initialinvestigations of Raman-based hormone detection are performed usingvasopressin-targeting aptamers reported in He et al. (Analytica ChimicaActa 759 (2013) 74-80). Since vasopressin is a nine amino acid hormone,like oxytocin, and differs from oxytocin by two amino acids, it is anexcellent proxy for oxytocin. The vasopressin aptamer was modified witha TAMRA-modified uracil phorphoramidite, to allow the changes in thestructure of the aptamer upon exposure to the target hormone to beidentified: TAMRA is known to have intense Raman spectral features thatcompare favorably with those of FAM, and has been used in similar Ramanbased detection methods for vasopressin. The TAMRA-modified base wasinserted at position 22 on the aptamer, the position farthest from theNP surface, which was hypothesized to produce the largest difference inTAMRA signal intensity upon exposure of the aptamer modified NPs tovasopressin.

The response of the vasopressin aptamer-modified NP were investigated intwo ways. The first established the ability of aptamer-based assays todifferentiate between target molecules through comparison of the changesobserved upon exposure of the system to vasopressin with those observedwhen the system is exposed to oxytocin and substance P, a neuropeptidethat is similar in length, size and mass, but not in sequence, tovasopressin. The second study established the effect of surfacemodification through comparison of the response observed using NPs withmonolayers composed of the dye-modified aptamer alone, and the aptamerwith MCP or MCH.

A comparison of the responses to the three proteins reveals theselectivity of the vasopressin-aptamer based SERS assay for the targetmolecule (FIG. 4, Panel A). Exposure of the aptamer-modified NPs toconcentrations of vasopressin over a range of 1 nM to 10 μM revealed alinear increase in intensity of the 1355 cm⁻¹ feature in the Ramanspectrum. This was not the case when the response was measured inresponse to exposure to a similar range of concentrations of oxytocin,where there was little correlation between the intensity of the selectedfeature and the concentration of the analyte. Exposure of the system tosubstance P revealed some correlation between concentration and featureintensity, but the correlation was not as good as that observed with thevasopressin. Surprisingly, the presence of MCP, which was intended as aninternal standard against which the intensity of the Raman signal couldbe quantified, significantly increases the intensity of the spectralfeatures (FIG. 4, Panel B). This effect, enhancement upon addition of asecond compound to the monolayer, has been observed upon addition ofMCH. However, addition of MCH in this setting did not increase theintensity of the spectral features, and dampened the responsecharacteristics, with no correlation between VP concentration andfeature intensity.

Beyond differentiation of structurally similar hormone molecules such asoxytocin and vasopressin using aptamer modified NPs, our efforts focusedon improving reproducibility of spectral feature intensities, a problemthat is a common to Raman-based analytical applications. Reproducibilityissues, particularly with dry samples, are often associated with thepresence of “hot spots”, or regions where analytes lay at the junctionof two Raman active NPs resulting in unusually large signal enhancementin a target for which the aptamer had been developed.

References for Examples 1-4

-   (1) Hoon, S. et al. BioTechniques 2011, 51, 413.-   (2) Stoltenburg, R. et al. J Anal Methods Chem 2012, 2012, 14.-   (3) Schütze, T. et al. PLoS ONE 2011, 6, e29604.-   (4) Latulippe, D. R. et al. Anal. Chem. 2013, 85, 3417.-   (5) Leona, M. Proc. Natl. Acad. Sci. 2009, 106, 14757.-   (6) O'Donnell, D. et al. Submitt. Publ. 2013.-   (7) Navarro, J. R. G. et al. Analyst 2013, 138, 583.-   (8) Liu, J. et al. Nat. Protoc. 2006, 1, 246.-   (9) Thompson, D. G. et al. Anal. Chem. 2008, 80, 272805.-   (10) Dougan, J. A. et al. Nucleic Acids Res. 2007, 35, 3668.-   (11) Hurst, S. J. et al. Anal. Chem. 2006, 78, 8313.-   (12) Cao, Y. C. et al. Science 2002, 297, 1536.-   (13) Yang, J. et al. ACS Nano 2013, 7, 5350.

II. REAGENTS AND METHODS FOR DETECTING LYME DISEASE (Examples 5-8)

Lyme disease is caused by a variety of tick-borne pathogens, such as thegram-negative spirochete Borrelia burgdorferi and transmitted by Ixodidtick species. It is the leading vector-borne infectious disease in theUnited States, with a steady rise in number of cases reported each year.(CDC—Cases by State—Lyme Diseasehttp://www.cdc.gov/lyme/stats/chartstables/reportedcases_statelocality.html(accessed Nov. 18, 2013)). While Lyme disease symptom presentation ofthe classic bull's-eye rash (erythema migrans or EM rash) in endemicareas indicates immediate treatment without accompanying diagnostictesting, as many as 20% of Lyme disease cases proceed to less welldiagnosed secondary symptoms without presentation or recognition of theEM rash. (Biesiada, G. et al. Arch Med Sci 2012, 8, 978).

The current CDC-approved diagnostic test is a two-tier system,consisting of an enzyme-linked immunosorbent assay (ELISA), followed byimmunoblot (IB) analysis. (Ellis, D. I. et al. Analyst 2013, 138, 3871;Wu, X. et al. Analyst 2013, 138, 3005). The second tier of testing, IBanalysis, is costly, time-consuming and technically challenging. Theinherent flaws in the two-tiered serological Lyme testing regime includethe complexity of interpreting the results and the dependence ofantibody production on timing post infection (DeBiasi, R. L. et al.Current Infectious Disease Reports 2014, 16, 450). If the tests aregiven either too early (6-8 weeks post infection) or too late (4-6months post infection), anti-Lyme antibodies may not be present atdetectable levels. This is especially problematic for Lyme disease,where symptoms such as fever, joint pain, and “brain fog” arenon-specific and difficult to diagnose. The clinical community feels thetest is so unreliable that primary care physicians are reluctant toprescribe it, many feeling their experience with the disease andinteraction with the patient is a more reliable diagnostic approach.This is a significant clinical shortcoming: accurate diagnosis of Lymedisease would allow its treatment with a short course of simpleantibiotics and would prevent the costs and suffering associated withuntreated cases. While numerous serological Lyme disease tests have beenrecently developed and are in use in the US, the new tests exhibitsimilar levels of false positives and/or additional false negatives dueto insufficient antigen presentation; (Wu, X. et al. Analyst 2013, 138,3005) none have replaced the established, CDC-approved two-tier testingformat.

Low sensitivity has plagued the CDC-approved test, a flaw that has notbeen addressed in tests promoted as improvements over the currentprotocol. Irrespective of whether the low sensitivity of the assaysystem is due to the immune-suppressing nature of the Borreliaspirochete, agents secreted by the tick vector, or other unknownfactors, it is clear a highly sensitive assay to detect the Lymepathogen, B. burgdorferi, and other tick-borne pathogens, in human bloodor serum would provide the frontline clinician with an invaluable toolin fighting this disease, a tool that is currently unavailable but isdesperately needed.

Example 5 Aptamer Selection for B. burgdorferi Surface Protein OspA

Novel, high-affinity Lyme specific aptamers were generated by employingspecially designed and fabricated micro-columns (FIG. 7, Panel A). Thesecolumns were packed with target conjugated-agarose beads, or blankagarose beads (for negative selection). The columns can be run in seriesor in parallel, enabling efficient selection of aptamers againstmultiple targets simultaneously. The schematic shown in FIG. 7, Panel Billustrates the selection of RNA aptamers, though the system is readilyadaptable for the generation of single-stranded DNA aptamers, simply byremoving the reverse transcription and transcription steps.

The aptamer development process utilized a streamlined aptamer selectionprocess called RNA Aptamer Isolation via Dual-cycles-SELEX(RAPID-SELEX), which eliminates unnecessary DNA amplification andpurification steps. (Szeto, K. et al. PLoS ONE 2013, 8, e82667.)RAPID-SELEX allows aptamer pools eluted from one set of columns to beadded to a second set of columns without DNA amplification betweenselection rounds, eliminating PCR amplification following every oddnumbered round. (Szeto, K. et al. PLoS ONE 2013, 8, e82667.) Eliminationof every other amplification and purification step reduces the averagetime for each round of aptamer selection by approximately 50%, and thetime required for the entire selection process by 67% to 75%.

High-throughput, Next Generation DNA Sequencing (NGS) was used todetermine the selected aptamer sequences from each round of microcolumnSELEX in conjunction with the Biotechnology Resource Center (BRC) andthe Bioinformatics Center of Cornell University. Illumina NGSinstruments were capable of generating 2×10⁷-2×10⁸ DNA sequences/run,facilitating highly accurate statistical analysis of enrichment of DNAsequences by the SELEX process.

Using the methods described above, DNA aptamers that bind and detectOspA were selected: SEQ ID NOs: 67-72.

TABLE 2 OspA aptamers OspA-21CATGACACCGTACCTGCTCTAATAAGCACGCCAGGGACTATTAGATCGGAATAGCACACGTCTGAACTCCAAGCACGCCAGGGACTATTA (SEQ ID NO: 67) OspA-46CATGACACCGTACCTGCTCTAATAAGCACGCCAGGGACTATTAGATCGGAAGAGCACACGTGTGAACTCCAAGCACGCCAGGGACTATTA (SEQ ID NO: 68) OspA-22CATGACACCGTACCTGCTCTACGAGATTCAAGCACTCCAGGGACTATTAGATCGGAAGAGCACACGTCTGAAGCACGCCAGGGACTATTA (SEQ ID NO: 69) OspA-39CATGACACCGTACCTGCTCTACGAGATTCAAGCACGCCAGGGATTATTAGATCGGAAGAGCACACGTCTGAAGCACGCCAGGGACTATTA (SEQ ID NO: 70) OspA-55CATGACACCGTACCTGCTCTTGCTTTTCGTGCGCGCATAAAATACTTTGATACTGTGCCGGATGAAAGCGAAGCACGCCAGGGACTATTA (SEQ ID NO: 71) OspA-59CATGACACCGTACCTGCTCTTGCTTTTCGTGCGCGCATAAAATACCTTGATACTGTGCCGTATGAAAGCGAAGCACGCCAGGGACTATTA (SEQ ID NO: 72)

Fluorescence anisotropy was used to determine the sensitivity of theaptamers generated for Osp A. FIG. 8 shows the results of fluorescenceanisotropy experiments demonstrating the binding of OspA aptamer to OspAprotein. The marked increase in anisotropy with increased target proteinconcentrations revealed that this OspA DNA aptamer had a K_(d)=2.2 nMfor OspA protein.

Aptamers specific to B. burgdorferi OspC and BmpA are generated,sequenced, and characterized using the same methods.

TABLE 3 OspC and BmpA aptamers OspC-CATGACACCGTACCTGCTCTGCGGTGCTGTATCGTCGTTTAGGCTGTTACCAGGGCC 23ACCGGACAGAGGTAAGCACGCCAGGGACTATTA (SEQ ID NO: 73) OspC-CATGACACCGTACCTGCTCTCGTATAGATCCTCTCGCGCTTCGGTTTTTAGAAGTATT 28CAAGGTATCATCAAGCACGCCAGGGACTATTA (SEQ ID NO: 74) OspC-CATGACACCGTACCTGCTCTGATCAGCCTGGTCAACGGGTGGTCCTGTGCCAAGCTC 30GAAAATTCGCCGAAAGCACGCCAGGGACTATTA (SEQ ID NO: 75) OspC-CATGACACCGTACCTGCTCTTGGAGCTAGAGAGCCGGTGATCGAAATTCTGGATGTT 34TCTGACGTTTGCTAAGCACGCCAGGGACTATTA (SEQ ID NO: 76) OspC-CATGACACCGTACCTGCTCTACCCCGGAAATGATTAGCCATTGTGGTACTCATCTGG 36GCAGTCAGCACATAAGCACGCCAGGGACTATTA (SEQ ID NO: 77) OspC-CATGACACCGTACCTGCTCTTTAACCCCTCGCGGAGGTGTACACGGGCCTACATAAT 37CCTCCGAGGTTCCAAGCACGCCAGGGACTATTA (SEQ ID NO: 78) BmpA-CATGACACCGTACCTGCTCTTTACGTTTGGGACGTCTGGCGAAGCCACCACAAGCTA 5GCCCTCCAATTTAAAGCACGCCAGGGACTATTA (SEQ ID NO: 79) BmpA-CATGACACCGTACCTGCTCTTTGATCATCACGGCACACTCATTACGGTTGGATATAC 6TAGTCCGGTTAGAAAGCACGCCAGGGACTATTA (SEQ ID NO: 80) BmpA-CATGACACCGTACCTGCTCTCCCTTCTGACTGGATGCCGGATCTGGGCCGATTTTGTT 7CGCGCCCCGCCCAAGCACGCCAGGGACTATTA (SEQ ID NO: 81) BmpA-CATGACACCGTACCTGCTCTTTCCGCTGGTTCCACGTGGTCCCGCGTAGGTTCGTGTG 8CGCGCAAAATCCAAGCACGCCAGGGACTATTA (SEQ ID NO: 82) BmpA-CATGACACCGTACCTGCTCTGCCCCTGCGTGCCGCAGTCAATCACCATGTTGTTATTA 9CGGACTACCTGGAAGCACGCCAGGGACTATTA (SEQ ID NO: 83) BmpA-CATGACACCGTACCTGCTCTCCGGTACGATAGGGGTTGAGTTGGACACACTGCCTGG 10TTAAATTGTGCAGAAGCACGCCAGGGACTATTA (SEQ ID NO: 84)

Fluorescence anisotropy can be complemented with EMSA and microplatecapture assays to allow binding affinity of DNA aptamers for theirtargets.

Example 6 Characterization of OspA, OspC and BmpA Aptamer Binding toTarget Proteins

Methods necessary to detect and quantify the binding affinity ofindividual aptamers to specific B. burgdorferi proteins are developed. Avariety of method can be used to demonstrate

target binding to potential DNA aptamer oligonucleotides. Whileelectrophoretic mobility shift assay (EMSA); fluorescence anisotropymeasurements; DNA pull-down assays; microplate capture assays can beused to determine aptamer affinity for its targets, the inventorseffectively employed fluorescence anisotropy to determine thesensitivity of aptamers generated for OspA (FIG. 8). Briefly,fluorescently labeled target protein, in this case either OspA or OspCwere assayed against increasing concentration of target specificaptamer. Changes in the rotational speed of the fluorescent proteinsupon aptamer binding were detected through changes in the polarizabilityof the dye emissions, providing a rapid and reliable test to determinethe affinity of the aptamer for its target. The marked increase inanisotropy with increased target protein concentrations revealed theOspA aptamer to bind with a K_(d) of approximately 2.2 nM.

Anisotropy measurements of fluorescently labelled proteins are mosteffective when aptamer binding significantly increases the size of thecomplex, resulting in a significant decrease in the rate of rotation ofthe aptamer in solution. While fluorescence anisotropy employedeffectively to determine the K_(d) of the OspA aptamer for its target,this is not true for all aptamer-target interactions. Shouldfluorescence anisotropy not prove effective, EMSA and microplate captureassays to allow binding affinity of DNA aptamers for their targets canbe utilized.

To identify aptamers with the highest affinity for the three targetproteins, up to six of the most commonly aptamer sequences for each B.burgdorferi protein are synthesized and screened. Concentrations rangingfrom 0.1 nM to 100 nM Lyme-specific aptamer are used for theseexperiments; binding affinities of <1 nM to 5 nM are considered adequatefor use. The 1 or 2 aptamers with the highest affinities for theirspecific target are employed in the Examples 7 and 8.

Example 7 Detection of B. burgdorferi Proteins by SERS

Nanoparticles are modified with the thiolated aptamers (ApNPs) optimizedin Example 5 for capture and detection of B. burgdorferi-specific OspA,OspC, and BmpA proteins. The Ag nanoparticle are synthesized using asimple, established literature procedure involving reduction of a silvernitrate solution with sodium citrate under microwave irradiation (Leona,M. Proc. Natl. Acad. Sci. 2009, 106, 14757) or Au NPs purchased from acommercial source (e.g. Ted Pella; Redding, Calif.; or Nanopartz;Loveland, Co.).

Surface modification with the aptamers is executed through standardthiol-metal interactions, and the concentration of aptamers appropriatefor the target NP size are calculated using Eqn. 1. (Taton, T. A. InCurrent Protocols in Nucleic Acid Chemistry; John Wiley & Sons Inc.: NewYork, 2002).

Amt of aptamer=A _(n) ×C _(n) ×D _(o) ×V  (1)

A_(n)=NP surface area (cm⁻²); C_(n)=particle conc. (particles L⁻¹);D_(o)=Ap density (mol cm⁻²); V=reaction vol. (L)

To determine the efficiency of surface modification,fluorophore-modified Lyme-specific aptamers are employed in thefluorometric quantitation technique developed by Mirkin and co-workers.(Demers, L. M. et al. Anal Chem 2000, 72, 5535).

The aptamers developed in Example 5 are modified with Raman-active dyesthat served as proxies for the target molecule. This is schematicallyrepresented in FIG. 9, Panel A. (figure derived from: Baker, B. R. etal. J. Am. Chem. Soc. 2006, 128, 3138) To demonstrate the principle, anaptamer developed for targeting vasopressin was modified with TAMRA, amolecule known to have a strong Raman signal (FIG. 10). (Cao, Y. C., etal. Science 2002, 297, 1536). The intensity of the Raman signalincreased in concert with the concentration of vasopressin,demonstrating the principle of Raman active dyes being used as proxiesfor the target molecule. Exposure of this TAMRA-modified aptamer tosimilar polypeptides (i.e. substance P and oxytocin; data not shown) notresult in a significant increase in Raman signal intensity,demonstrating that the aptamer-based SERS-detection approach was capableof both detecting its target and differentiating the target frommolecules with significant structural similarity.

While FIG. 9, Panel B demonstrates the principle of dye-modifiedaptamers as recognition elements, the placement of the dye in thesequence, and the identity of the dye can provide optimal sensitivityfor the Lyme disease-related proteins. Each aptamer developed in Example5 is modified with a Raman-active marker in the terminal position and atmultiple internal positions. The dyes are selected for high signalintensity (e.g., FAM, Cy3, Cy 3.5, Cy5 and TAMRA), and distinct spectralfeatures that allowed them to easily be identified (FIG. 10). (Cao, Y.C. et al. J. Am. Chem. Soc. 2002, 125, 14676). The Raman-active dyes areincorporated into the aptamer DNA sequences during oligonucleotidesynthesis using commercial reagents.

Raman investigations are initiated using research grade instrumentationto optimize the spectroscopic method. These methods are then bere-tested using a portable, commercially available spectrometer using785 nm incident light, a wavelength frequently used in portablecommercial bench top or handheld Raman spectrometers.

Each of the aptamer-modified NPs is exposed to a solution containing oneof the B. burgdorferi-specific proteins for which it is designed,allowing the aptamer to capture the target. This process is repeatedwith each of the ApNPs in the presence of the B. burgdorferi proteinsfor which the aptamer is not developed, to demonstrate specificity ofthe ApNPs for their target molecules. To optimize incubation conditionsfor target capture, three variables are investigated: aptamerconcentration at the NP surface, concentration and composition ofadditives intended to stabilize and optimize Raman spectral features,and incubation time of the ApNPs with the target metabolites. Theoptimal combination of aptamer concentration at the NP surface, andincubation time are investigated to determine the limit of detection foreach of the analytes. The limit of detection established in theseinvestigations are compared with the sensitivity of established standardbiological assays, such as commercial Lyme immunoassays (e.g. ELISA),providing a value against which subsequent investigations can becompared.

Other surface modifications may be performed. For example the surfacemodifications may include PEG to coat parts of the nanoparticle surfacenot covered by aptamers. Alternatively, the surface modifications mayinclude alkyl thiols.

Example 8 Rapid Detection of B. burgdorferi Proteins Human Lyme DiseaseSerum Panels

Rapid detection of B. burgdorferi proteins in human Lyme disease serumpanels is performed in collaboration with Clinical and TranslationalScience Center and the Joint Clinical Trials Office at Weill CornellMedical College. Human serum panels are obtained from two separatesources: a commercially available Lyme disease serum panel fromSeraCare, a company that provides human serum panels for a variety ofinfectious and other disease states (www.seracare.com/Products/Panels);and Lyme disease serum panels available from the US CDC (Molins, 2014doi:10.1128/JCM.01409-14). These panels include serum from uninfectedindividuals and from individuals infected with B. burgdorferi at variousstages of disease. These panels are ideal for pre-clinical testing.

For these studies, 5-10 μL of human serum, either negative control orLyme positive samples are diluted into buffer and mixed with ApNPs asdescribed in Example 6. Following binding of the ApNPs to the B.burgdorferi target proteins, the samples are divided in half; onealiquot is analyzed for SERS in a bench top Raman spectrometer, and thesecond is reserved for analysis using commercial B. burgdorferiimmunoassays. The panels referenced above include serum from uninfectedindividuals and are used as negative controls. In these studies, anumber of variables are directly studied, including: the time requiredfor optimum binding; the concentration of ApNPs required for optimalsignal; minimal amount of human serum necessary for detection of the B.burgdorferi proteins; and the relative sensitivities of the OspA, OspC,and BmpA assays. The sensitivity and specificity of the SERS-based B.burgdorferi assays are determined. A variety of statistical analyses areused to determine the best diagnostic models and cut-off required tooptimize B. burgdorferi detection in humans.

Rapid Detection of B. burgdorferi Proteins from Infected Ixodesscapularis Ticks

Assays developed in Examples 5, 6, and 7 are used to demonstrate thedetection of B. burgdorferi in infected I. scapularis ticks. Ticksobtained from two separate sources are used. Control, non-infected I.scapularis tick nymphs are purchased from the Oklahoma State UniversityTick Rearing Facility. The Tick Rearing facility is a fee-for-servicefacility that provides uninfected, laboratory raised ticks at variouslife cycle stages for research applications.

Wild B. burgdorferi-infected ticks are collected in collaboration withthe Department of Natural Resources at Cornell University. The effectsof environmental conditions on the survival of I. scapularis in theirhabitat are studied. Ticks are collected as follows: Ixodes scapularisnymphs are collected from the field between May and July during theirseasonal activity peak. (Falco, R. C. et al. Am. J. Epidemiol. 1999,149, 771) A well-established drag sampling method is used to collectthem: a white cloth with an area of one square meter is dragged overvegetation and leaf litter in forested areas. (Schulze, T. L. et al. J.Med. Entomol. 1997, 34, 615.) The cloth mimics the animal hair fibers towhich I. scapularis attaches itself. Nymphs are stored at −80° C. at theCary Institute until they can be used for further analysis. Studies fromthe Cary Institute show that as many as 55% of I. scapularis nymphscollected in the Hudson River Valley of New York are infected with B.burgdorferi. (Aliota, M. T. et al. Vector Borne Zoonotic Dis. Larchmt. N2014, 14, 245)

Between 5 and 10 tick nymphs, either lab reared or collected, are lysedby placing them in a micro centrifuge tube and homogenized with adisposable homogenizer in buffered lysing solution. Once the ticks areruptured, aliquots of the homogenate are briefly centrifuged to removedebris and the extract is mixed with ApNPs as described in Example 7.Following binding of the ApNPs to the B. burgdorferi target proteins,the samples are divided in half; one aliquot is analyzed for SERS in abench top Raman spectrometer, and the second is reserved for analysisusing commercial B. burgdorferi immunoassays. Lab raised tick nymphsobtained from the Oklahoma State University Tick Rearing Facility serveas negative controls.

A number of variables are directly studied, including: the time requiredfor optimum binding; the concentration of ApNPs required for optimalsignal; minimal number of infected nymphs necessary for detection of theOsp proteins; and the relative sensitivities of the OspA, OspB, and OspCassays. The sensitivity and specificity of the SERS-based B. burgdorferiassays are determined.

INCORPORATION BY REFERENCE

The contents of all references, patent applications, patents, andpublished patent applications, as well as the Figures and the SequenceListing, cited throughout this application are hereby incorporated byreference in their entirety as if each individual publication or patentwas specifically and individually incorporated by reference. In case ofconflict, the present application, including any definitions herein, maycontrol.

EQUIVALENTS

It will be understood by those skilled in the art that various changesin form and details may be made therein without departing from thespirit and scope of the invention as set forth in the appended claims.Those skilled in the art will recognize, or be able to ascertain usingno more than routine experimentation, many equivalents to the specificembodiments of the invention described herein. While specificembodiments of the subject invention have been discussed, the abovespecification is illustrative and not restrictive. Many variations ofthe invention may become apparent to those skilled in the art uponreview of this specification. The full scope of the invention should bedetermined by reference to the claims, along with their full scope ofequivalents, and the specification, along with such variations. Suchequivalents are intended to be encompassed by the following claims.

What is claimed:
 1. A surface enhanced Raman spectroscopy (SERS)-active reagent for detecting an analyte comprising: (a) one or more SERS-active surface; (b) unmodified or modified with one or more aptamer; and (c) one or more Raman-active marker.
 2. The reagent according to claim 1, wherein the SERS-active surface is selected from the group consisting of metals (including but not limited to silver, gold, copper, certain other transition metals and titanium nitride) semiconductor substrates (including but not limited to titanium oxide, zinc oxide, zinc selenide) or semimetals (including but not limited to graphene and molybdenum disulfide).
 3. The reagent according to claim 1, wherein the aptamer is functionalized.
 4. The reagent according to claim 3, wherein the aptamer is covalently or non-covalently attached to the SERS-active surface.
 5. The reagent according to claim 1, wherein the aptamer comprises a nucleotide sequence selected from the group consisting of SEQ ID NOs: 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, and
 84. 6. The reagent according to claim 1, wherein the Raman-active marker comprises a dye, fluorescent marker, carbon nanotubes, fullerenes, alkene, alkyne, or azide.
 7. The reagent according to claim 6, wherein the Raman-active marker is a fluorescent or non-fluorescent Raman-sensitive marker, and said marker is selected from the group consisting of azides, alkynes, fluorescein (FAM), Carboxytetramethylrhodamine (TAMRA), Cy3, Texas-Red (TR), Cy3.5, Rhodamine 6G, Cy5, TRIT (tetramethyl rhodamine isothiol), NBD (7-nitrobenz-2-oxa-1,3-diazole), phthalic acid, terephthalic acid, isophthalic acid, cresyl fast violet, cresyl blue violet, brilliant cresyl blue, para-aminobenzoic acid, erythrosine, biotin, digoxigenin, 5-carboxy-4′,5′-dichloro-2′,7′-dimethoxy fluorescein, 5-carboxy-2′,4′,5′,7′-tetrachlorofluorescein, 5-carboxyfluorescein, 5-carboxy rhodamine, 6-carboxyrhodamine, 6-carboxytetramethyl amino phthalocyanines, azomethines, cyanines, xanthines, succinylfluoresceins, aminoacridine, and quantum dots.
 8. The reagent according to claim 1, wherein the aptamer undergoes a conformational change upon binding of the analyte.
 9. The reagent according to claim 8, wherein the conformational change brings the Raman-active marker into close proximity to the surface of the SERS-active surface and leads to an enhancement in the Raman signal.
 10. The reagent according to claim 1, wherein the Raman-active marker is covalently attached to the aptamer.
 11. The reagent according to claim 1, wherein the analyte is selected from the group consisting of amino acid, peptide, polypeptide, protein, glycoprotein, lipoprotein, nucleoside, nucleotide, oligonucleotide, nucleic acid, sugar, carbohydrate, oligosaccharide, polysaccharide, fatty acid, lipid, hormone, metabolite, cytokine, chemokine, receptor, neurotransmitter, antigen, allergen, antibody, substrate, metabolite, cofactor, inhibitor, drug, pharmaceutical, nutrient, prion, toxin, poison, explosive, pesticide, chemical warfare agent, biohazardous agent, radioisotope, vitamin, heterocyclic aromatic compound, carcinogen, mutagen, narcotic, amphetamine, barbiturate, hallucinogen, waste product, and contaminant.
 12. A diagnostic kit comprising: a) the SERS-active reagent of claim 1; b) at least one Raman-sensitive marker control c) at least one positive biological fluid or tissue control; and c) at least one negative biological fluid or tissue control.
 13. A detection system comprising: a) the SERS-active reagent of claim 1; and b) a Raman detector.
 14. The system according to claim 13, wherein the Raman detector is portable or not portable.
 15. The system according to claim 13, further comprising a sample collection apparatus.
 16. A method for determining the presence of one or more analyte in a biological sample, the method comprising: a) receiving a biological sample; b) contacting the biological sample to at least one SERS-active reagent comprising: (i) one or more SERS-active surface; (ii) unmodified or modified with one or more aptamer; and (iii) one or more Raman-active marker; c) allowing the analyte to come into contact with the aptamer; d) binding of the analyte to the aptamer, wherein said binding causes the aptamer to undergo a conformational change; e) irradiating the at least one SERS-active reagent bound to the one or more analyte; f) detecting the Raman signal to generate (a) Raman spectra (um); and g) comparing the Raman signal detected in (f) with a reference Raman signal of a control, wherein the presence of one or more analyte in the biological sample is determined when said Raman signal detected in (f) differs from said reference Raman signal.
 17. The method of claim 16, wherein an increase in the Raman signal in the Raman spectra in (f) compared to control can be correlated with the amount of the one or more analyte.
 18. The method of claim 17, wherein the conformational change in the aptamer upon binding of the analyte brings the Raman-active marker into close proximity of the SERS-active surface.
 19. A method for diagnosing a disease or disorder in a subject comprising the steps of: a) receiving a biological sample from a subject; b) contacting the biological sample to at least one SERS-active reagent comprising: (i) one or more SERS-active surface; (ii) unmodified or modified with one or more aptamer; and (iii) one or more Raman-active marker; c) allowing binding of the at least one SERS reagent to one or more analyte in the biological sample, wherein said binding causes a conformational change to the one or more aptamer of the SERS reagent; d) irradiating the at least one SERS-active reagent bound to the one or more analyte; e) detecting the Raman signal of the at least one SERS-active reagent; and f) comparing the Raman signal of said at least one SERS-active reagent detected in (e) with a reference Raman signal of said at least one SERS-active reagent detected in a laboratory derived negative control sample, biological sample received from a control subject (healthy subject), or pooled biological samples from numerous control subjects (healthy subjects) wherein said disease is diagnosed when said Raman signal detected in (e) differs from said reference Raman signal in position and/or intensity.
 20. The method of claim 19, wherein the disease or disorder is selected from the group consisting of an infectious disease, proliferative disease, neurodegenerative disease, cancer, psychological disease, metabolic disease, autoimmune disease, sexually transmitted disease, gastro-intestinal disease, pulmonary disease, cardiovascular disease, stress- and fatigue-related disorder, fungal disease, pathogenic disease, and obesity-related disorder. 